Gene targeting using replicating DNA molecules

ABSTRACT

The invention provides novel methods of gene targeting using replication in order to increase the efficiency of targeted genetic modification in an eukaryotic organism. Included are vectors, expression cassettes, and modified cells, plants and seeds.

TECHNICAL FIELD

[0001] The present invention relates generally to plant molecularbiology.

SUMMARY OF THE INVENTION

[0002] The present invention provides novel methods and compositions forcarrying out gene targeting. The method uses homologous recombinationprocesses endogenous in the cells of all organisms. Any gene of anyorganism can be modified by the methods of the invention as long as thesequence of at least a portion of the gene is known, or a DNA clone isavailable.

[0003] The invention provides methods for increasing gene targetingfrequencies comprising introducing into a target cell a targeting vectorcomprising an origin of replication and further comprising a targetmodifying sequence which is compatible with a target site in the genomeof the target cell. The target modifying sequence comprises the sequencemodifications to be introduced into the target site sequence. Areplicase is also provided in the target cell. Replication of thetargeting vector stimulates homologous recombination between thetargeting vector and the target site resulting in a gene targetingevent.

[0004] In another embodiment, the invention provides methods forincreasing gene targeting frequencies comprising introducing into atarget cell a targeting vector comprising a transposon comprising anorigin of replication and further comprising a target modifying sequencewhich is compatible with a target site in the genome of the target cell.A transposase is provided in the target cell, wherein the transposase iscapable of excising the transposon to produce a replication-competenttargeting vector. A replicase is also provided in the target cell.Replication of the targeting vector stimulates homologous recombinationbetween the targeting vector and the target site resulting in a genetargeting event.

[0005] In another embodiment the invention provides methods forincreasing gene targeting frequencies comprising introducing into atarget cell a targeting vector comprising an origin of replication andfurther comprising a target modifying sequence which is compatible witha target site in the genome of the target cell, wherein the origin ofreplication and the target modifying sequence are flanked bysite-specific recombination sites. A site-specific recombinase capableof excising the targeting vector to produce a replication-competenttargeting vector is provided. A replicase is also provided in the targetcell. Replication of the targeting vector stimulates homologousrecombination between the targeting vector and the target site resultingin a gene targeting event.

[0006] The invention also provides cells and organisms produced by themethods. These cells or organisms comprise a modified targetpolynucleotide sequence produced by a method of the invention. Theinvention further provides progeny or seed produced by the modifiedcells or organisms, wherein the progeny or seed have inherited the genetargeted modification. The invention also provides isolated nucleicacids such as targeting vectors.

[0007] The compositions used in the invention comprise nucleic acids,such as targeting vectors, and expression cassettes. The compositionsfurther comprise donor organisms comprising an integrated targetingvector, and target organisms comprising modified target sequences, andthe progeny of each.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Definitions

[0009] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in its naturally occurring environment or (2) if the material isin its natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in the cellother than the locus native to the material.

[0010] As used herein, “polypeptide” and “protein” are usedinterchangeably and mean proteins, protein fragments, modified proteins,amino acid sequences and synthetic amino acid sequences. The polypeptidecan be glycosylated or not.

[0011] As used here, “polynucleotide” and “nucleic acid” are usedinterchangeably. A polynucleotide can be full-length or a fragment andincludes polynucleotides that have been modified for stability. Unlessotherwise indicated, the term includes reference to a specific sequenceor its complement.

[0012] As used herein, “functional variant” or “functional derivative”or “functional fragment” are used interchangeably. As applied topolypeptides, the functional variant or derivative is a fragment, amodified polypeptide, or a synthetic polypeptide that provides afunctional activity in a manner similar to the wild type, or naturallyoccurring, gene products

[0013] As used herein, “origin of replication” refers to apolynucleotide region where DNA replication is initiated. The origin ofreplication is intended to include functional fragments, modifications,variants, and derivatives which retain the functional activity.Replication is usually initiated at the origin of replication by areplicase polypeptide.

[0014] As used herein, “replicase”, or “replicase polypeptide” refers topolypeptides capable of stimulating DNA synthesis. The polynucleotidesand polypeptides are intended to include functional variants, fragments,and derivatives which retain the functional activity. The polypeptidesinclude proteins commonly referred to as “replication proteins”,“replication associated proteins”, or “replication initiation proteins”.The polypeptide includes proteins in which all the “replicationassociated” or “replication” functions are encoded as a single protein,and those in which these functions are carried out by more than oneprotein, irrespective of whether proper or “inappropriate” splicing hasoccurred prior to translation.

[0015] As used herein, “replicase polynucleotide” refers topolynucleotides coding for a replicase polypeptide, including functionalvariants, derivatives, fragments, or functional homologs ofcharacterized replicase polynucleotides. Replicase polynucleotides,functional variants and/or functional homologs from any organism can beused in the methods of the invention as long as the expressed replicasepolypeptides bind to the origin of replication, and/or stimulate DNAreplication.

[0016] As used herein, “plant” includes but is not limited to wholeplants, plant parts, plant cells, plant tissue, and plant seeds.

[0017] As used herein, “site-specific recombinase” refers to any enzymecapable of being functionally expressed that catalyzes conservativesite-specific recombination between its corresponding site-specificrecombination sites. The site-specific recombinase may be naturallyoccurring, or a recombinantly produced polypeptide, fragment, variant,or derivative thereof that retains the activity of the naturallyoccurring recombinase.

[0018] As used herein “gene targeting” refers to a process whereby aspecific sequence modification is facilitated at a desired genetic locusby a transforming nucleic acid, such as a targeting vector. Typically,the gene at the target locus is modified, removed, replaced orduplicated by the transforming nucleic acid. Modifications include atleast one insertion, deletion, or substitution of one or morenucleotides at a target site.

[0019] As used herein “homologous recombination” refers to the processby which a recombination event occurs between two homologous nucleicacid regions.

[0020] As used herein “transposon” refers to a DNA sequence capable ofmoving from one place in the genome to another. Transposons aretypically characterized by being flanked by terminal inverted repeatsequences required for transposition.

[0021] As used herein “transposase” refers to a polypeptide thatmediates transposition of a transposon from one location in the genometo another. Transposases typically function to excise the transposon,and to recognize subterminal repeats and bring together the ends of theexcised transposon, in some systems other proteins are also required tobring together the ends during transposition.

[0022] As used herein “targeting vector” refers to a nucleic acidcomprising at least an origin of replication and a target modifyingpolynucleotide, wherein the target modifying polynucleotide comprises amodified version of the target sequence, containing any sequencemodification to be introduced at the target site resulted in a desiredgenetic change at the target. The targeting vector can be integrated ina host genome and later excised to produce a gene targeting event. Thetargeting vector can be provided by any transformation method orintroduced by sexual crossing.

[0023] As used herein “target polynucleotide” or “target site” refers toa polynucleotide sequence to be modified in the host organism. Thetarget polynucleotide can be either an endogenous polynucleotide, or anexogenous polynucleotide previously introduced into the host organism.The target sequence may be any polynucleotide sequence, including butnot limited to a polypeptide coding region. The target sequence may be anon-coding region, for example, a promoter, an intron, a terminator, anenhancer, or any other regulatory, structural polynucleotide, or otherpolynucleotide region.

[0024] As used herein “target modifying polynucleotide” refers to apolynucleotide comprising the sequence modification to be incorporatedat the target site, wherein the sequence modification comprises at leastone base pair difference as compared to the target site sequence.Sequence modifications to the target polynucleotide may includenucleotide substitutions, nucleotide or polynucleotide deletions, and/ornucleotide or polynucleotide insertions.

[0025] As used herein “donor organism” or “donor cell” refers to anorganism, or cell, which comprises at least one of the following: atargeting vector, a replicase expression cassette, a recombinaseexpression cassette, and/or a transposase expression cassette, such thatthose components contained can be delivered to a target host organism ina heritable manner. For example, the component(s) may be delivered bysexually crossing the target host to the donor organism.

[0026] As used herein “target organism”, “target cell”, “host organism”,or “host cell” refers to an organism, or cell, which comprises at leastone target polynucleotide to be modified. Any one of the following: atargeting vector, a target modifying polynucleotide, a replicaseexpression cassette, a recombinase expression cassette, and/or atransposase expression cassette, may be introduced by any meansincluding transient or stable transformation, sexually crossing to adonor, or fusion to a donor cell.

[0027] As used herein “expression cassette” refers to a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA,virus, or nucleic acid fragment. Typically, the expression cassetteportion of an expression vector includes, among other sequences, anucleic acid to be transcribed, and a promoter.

[0028] As used herein “operably linked” includes reference to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0029] In general, the invention provides a method for gene targeting inan organism by providing a targeting vector comprising a targetmodifying polynucleotide and an origin of replication, and providingreplicase activity such that targeted sequence modifications areincorporated at the target site in the host genome. The target site canbe any polynucleotide region, including but not limited to polypeptidecoding regions, introns, exons, untranslated regions (UTR's), promoters,enhancers, terminators, or other regulators of gene expression, or anyother region of interest. The targeting vector comprises at least anorigin of replication and a target modifying polynucleotide. The targetmodifying polynucleotide shares sufficient homology with the target siteso that homologous recombination can occur between the twopolynucleotides. The target modifying polynucleotide has at least onebase pair difference as compared to the target site, this base pairdifference can comprise a point mutation (base change), insertion, ordeletion. When replicase activity is provided, the frequency ofincorporation of the sequence modifications at the target site isenhanced. The target site modification includes any changes which couldsuppress gene expression, such as the introduction of a premature stopcodon, frameshift mutation, or changes to a promoter or other UTR, andthe like. The changes also include modifications to increase geneexpression or protein activity such as alterations to codons, oralterations to UTR's and the like. The targeting vector need not beintegrated into the host genome, but may be maintained as anautonomously replicating vector. The targeting vector need not becircular in order to replicate, as illustrated by the work of Jeske etal. (2001) EMBO 20:6158-6167. The targeting vector may be introduced byany method, depending on the organism, including Agrobacterium-mediatedtransformation, biolistic methods, direct DNA delivery methods includingmicroinjection, chemical methods, electroporation and the like.

[0030] The targeting vector may further comprise a replicase expressioncassette wherein a replicase polynucleotide is operably linked to apromoter and other regulatory elements needed for expression of areplicase polypeptide. The promoter can be constitutive, inducible, orunder developmental control as needed in order to regulate theexpression of the replicase polypeptide.

[0031] If the targeting vector is incorporated into the genome, a methodof excision can be used to release the targeting vector. The targetingvector may comprise flanking sequences for excision using systems suchas site-specific recombinases, or transposases. The recombinase ortransposase activity may be introduced using a recombinant expressioncassette wherein the recombinase or transposase is operably linked to apromoter and other regulatory elements needed for expression of thepolypeptide. The recombinase or transposase activity may also beprovided by crossing a donor organism, which comprises a recombinase ortransposase expression cassette, with a target organism comprising theintegrated targeting vector. Methods of providing the transposase bycrossing organisms are disclosed in WO 01/71019, the contents of whichare herein incorporated by reference.

[0032] Replication

[0033] Examples of replication systems suitable for this inventioninclude bacterial origins of replication and replication proteins, viralorigins of replication and replication proteins, and eukaryoticreplication systems.

[0034] Examples of suitable viral replication systems include abutilonmosaic virus (AbMV), African cassaya mosaic virus (ACMV), banana streakvirus (BSV), bean dwarf mosaic virus (BDMV), bean golden mosaic virus(BGMV), beet curly top virus (BCTV), beet western yellow virus (BWYV),and other luteoviruses, cassaya latent virus (CLV), carnation etchedring virus (CERV), cauliflower mosaic virus (CaMV), chloris striatemosaic virus (CSMV), commelina yellow mottle virus (CoYMV), cucumbermosaic virus (CMV), dahlia mosaic virus (DMV), digitaria streak virus(DSV), figwort mosaic virus (FMV), hop stunt viroid (HSV), maize streakvirus (MSV), mirabilias mosaic virus (MMV), miscanthus streak virus(MiSV), potato stunt tuber virus (PSTV), panicum streak virus (PSV),potato yellow mosaic virus (PYMV), rice tungro bacilliform virus (RTBV),soybean chlorotic mottle virus (SoyCMV), squash leaf curl virus (SqLCV),strawberry vein banding virus (SVBV), sugarcane streak virus (SSV),thistle mottle virus (ThMV), tobacco mosaic virus (TMV), tomato goldenmosaic virus (TGMV), tomato mottle virus (TmoV), tobacco ringspot virus(TobRV), tobacco yellow dwarf virus (TobYDV), tomato leaf curl virus(TLCV), tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curlvirus—Thailand (TYLCV-t), wheat dwarf virus (WDV), and the bean yellowdwarf virus (BYDV). Other plant viruses with DNA replicases suitable foruse in the invention include members of the nanovirus group such asbanana bunchy top virus (BBTV), milk vetch dwarf virus (MDV),subterranean clover stunt virus (SCSV), and Ageratum yellow vein virus(AYVV).

[0035] Other virus systems include the papova viruses such as SV40,polyoma viruses, adenoviruses, papillomaviruses such as humanpapillomavirus (HPV) and bovine papillomavirus (BPV), herpes virusessuch as herpes simplex virus (HSV), cytomegalovirus (CMV), andEpstein-Barr virus (EBV), and retroviruses such as humanimmunodeficiency virus (HIV), human T lymphotropic virus (HTVL), simianimmunodeficiency virus (SIV), simian sarcoma virus (SSV), Rous sarcomavirus (RSV), caprine arthritis-encephalitis virus (CAEV), murineleukemia virus (MLV), avian leukemia virus (ALV), bovine leukemia virus(BLV), feline immunodeficiency virus (FIV), equine infectious anemiavirus (EIAV), and endogenous retrovirus (ERV), or a baculovirus system.For example, a viral vector system for use in animal cells is disclosedin WO 99/09139, and herein incorporated by reference.

[0036] Excision of Integrated Targeting Vectors

[0037] The targeting vector, flanked by site-specific recombinationsites and/or transposon sequences, may be randomly integrated in thegenome of a donor or target organism. Gene targeting can be activated byexcising the target vector, which is then capable of replication andhomologous recombination with the target sequence. The integrated vectormay be excised by providing site-specific recombinase or a transposaseactivity. Any system or method to excise the integrated targeting vectorcan be used in the invention.

[0038] Examples of transposons and transposases suitable for thisinvention include the P element transposon from Drosophila (Gloor, G. B.et al. (1991) Science 253:1110-1117), the Copia, Mariner and Minoselements from Drosophila, the Hermes elements from the housefly, thePiggyBack elements from Trichplusia ni, Tc1 elements from C. elegans,the Ac/Ds, Dt/rdt, Mu-M1/Mn, and Spm(En)/dSpm elements from maize, theTam elements from snapdragon, the Mu transposon from bacteriophage,bacterial transposons (Tn) and insertion sequences (IS), Ty elements ofyeast (retrotransposon), Ta1 elements from Arabidopsis(retrotransposon), IAP elements from mice (retrotransposon), and thelike. A transposable element system effective in vertebrates andinvertebrates is a synthetic SB transposon system derived fromTc1/mariner disclosed in WO 98/40510, the contents of which is hereinincorporated by reference.

[0039] Site-specific recombination systems are reviewed in Sauer (1994)Current Opinion in Biotechnology 5:521-527, Nunes-Duby et al. (1998)Nucl. Acids Res. 26:391-406, and Sadowski (1993) FASEB 7:760-767, thecontents of which are herein incorporated by reference. Anysite-specific recombination can be used in the invention. Examples ofsite-specific recombination systems suitable for this invention includethe integrase family, such as the FLP/FRT system from yeast, and theCre/Lox system from bacteriophage P1, as well as the Int, and R systems.The resolvase family can also be used, for example γδ resolvase, and thelike. Examples of site-specific recombination systems used in plants canbe found in U.S. Pat. No. 5,929,301; U.S. Pat. No. 6,175,056; WO99/25821; U.S. Pat. No. 6,331,661; WO 99/25855; WO 99/25841, and WO99/25840, the contents of each are herein incorporated by reference.

[0040] Markers

[0041] Gene targeting can be performed without selection if there is asensitive method for identifying recombinants, for example if thetargeted gene modification can be easily detected by PCR analysis, or ifit results in a certain phenotype. However, in most cases,identification of gene targeting events will be facilitated by the useof markers. Markers useful in the invention include positive andnegative selectable markers as well as markers that facilitatescreening, such as visual markers. Selectable markers include genescarrying resistance to an antibiotic such as spectinomycin (e.g. theaada gene, Svab et al. 1990 Plant Mol. Biol. 14:197), streptomycin(e.g., aada, or SPT, Svab et al. 1990 Plant Mol. Biol. 14:197; Jones etal. 1987 Mol. Gen. Genet 210:86), kanamycin (e.g., nptII, Fraley et al.1983 PNAS 80:4803), hygromycin (e.g., HPT, Vanden Elzen et al. 1985Plant Mol. Biol. 5:299), gentamycin (Hayford et al. 1988 Plant Physiol.86:1216), phleomycin, zeocin, or bleomycin (Hille et al. 1986 Plant Mol.Biol. 7:171), or resistance to a herbicide such as phosphinothricin (bargene), or sulfonylurea (acetolactate synthase (ALS)) (Charest et al.(1990) Plant Cell Rep. 8:643), genes that fulfill a growth requirementon an incomplete media such as HIS3, LEU2, URA3, LYS2, and TRP1 genes inyeast, and other such genes known in the art. Negative selectablemarkers include cytosine deaminase (codA) (Stougaard 1993 Plant J.3:755-761), tms2 (DePicker et al. 1988 Plant Cell Rep. 7:63-66), nitratereductase (Nussame et al. 1991 Plant J. 1:267-274), SU1 (O'Keefe et al.1994 Plant Physiol. 105:473-482), aux-2 from the Ti plasmid ofAgrobacterium, and thymidine kinase. Screenable markers includefluorescent proteins such as green fluorescent protein (GFP) (Chalfie etal., 1994 Science 263:802; U.S. Pat. No. 6,146,826; U.S. Pat. No.5,491,084; and WO 97/41228), reporter enzymes such as β-glucuronidase(GUS) (Jefferson R. A. 1987 Plant Mol. Biol. Rep. 5:387; U.S. Pat. No.5,599,670; and U.S. Pat. No. 5,432,081), β-galactosidase (lacZ),alkaline phosphatase (AP), glutathione S-transferase (GST) andluciferase (U.S. Pat. No. 5,674,713; and Ow et al. 1986 Science234(4778):856-859), visual markers like anthocyanins such as CRC (Ludwiget al. (1990) Science 247(4841):449-450) R gene family (e.g. Lc, P, S),A, C, R-nj, body and/or eye color genes in Drosophila, coat color genesin mammalian systems, and others known in the art.

[0042] One or more markers may be used in order to select and screen forgene targeting events. One common strategy for gene disruption involvesusing a target modifying polynucleotide in which the target is disruptedby a promoterless selectable marker. Since the selectable marker lacks apromoter, random integration events are unlikely to lead totranscription of the gene. Gene targeting events will put the selectablemarker under control of the promoter for the target gene. Gene targetingevents are identified by selection for expression of the selectablemarker. Another common strategy utilizes a positive-negative selectionscheme. This scheme utilizes two selectable markers, one that confersresistance (R⁺) coupled with one that confers a sensitivity (S⁺), eachwith a promoter. When this polynucleotide is randomly inserted, theresulting phenotype is R⁺/S⁺. When a gene targeting event is generated,the two markers are uncoupled and the resulting phenotype is R⁺/S⁻.Examples of using positive-negative selection are found in Thykjaer etal. (1997) Plant Mol. Biol. 35:523-530; and WO 01/66717, which areherein incorporated by reference.

[0043] Target Sequences

[0044] The methods of the invention can be practiced in any organism inwhich a method of transformation is available, and for which there is atleast some sequence information for the target sequence of interest, orfor a region flanking the target sequence of interest. It is alsounderstood that two or more sequences could be targeted by sequentialtransformation, co-transformation with more than one targeting vector,or the construction of a targeting vector comprising more than onetarget modifying sequence.

[0045] The target sequences can be selected from any portion of a genomeof interest. Typically, targets comprise genes or regulatory regions,although regions adjacent to or near genes may be selected such thatmodifications may be made without disrupting gene expression.

[0046] General categories of target sequences of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins.

[0047] Target sequences further include coding regions and non-codingregions such as promoters, enhancers, terminators, introns and the like,which may be modified in order to alter the expression of a gene ofinterest. For example, an intron sequence can be added to the 5′ regionto increase the amount of mature message that accumulates (see forexample Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); and Calliset al., Genes Dev. 1:1183-1200 (1987)).

[0048] The target sequence may be an endogenous sequence, or may be anintroduced exogenous sequence, or transgene. For example, this methodmay be used to alter the regulation or expression of a transgene, or toremove a transgene or other introduced sequence such as an introducedsite-specific recombination site. A sequence of interest could also beintroduced at a target site, for example a site-specific recombinationsite could be introduced, a endonuclease restriction site could beintroduced, a polynucleotide tag could be introduced, or a proteinpurification tag such as that encoding hexa-histidine could be insertedto facilitate purification of a expressed protein of interest.

[0049] In plants, more specific categories of target sequences includegenes encoding agronomic traits, insect resistance, disease resistance,herbicide resistance, sterility, grain characteristics, and commercialproducts. Genes of interest also included those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting, forexample, kernel size, sucrose loading, and the like. The quality ofgrain is reflected in traits such as levels and types of oils, saturatedand unsaturated, quality and quantity of essential amino acids, andlevels of cellulose.

[0050] Herbicide resistance traits may include genes coding forresistance to herbicides that act to inhibit the action of acetolactatesynthase (ALS), in particular the sulfonylurea-type herbicides (e.g.,the acetolactate synthase (ALS) gene containing mutations leading tosuch resistance, in particular the S4 and/or Hra mutations). Glyphosatetolerance can be obtained form the EPSPS gene.

[0051] Sterility genes can also be targeted, including maletissue-preferred genes and genes with male sterility phenotypes such asQM, described in U.S. Pat. No. 5,583,210. Other genes include kinasesand those encoding compounds toxic to either male or femalegametophytes.

[0052] For example, in Arabidopsis, the TGA3 locus was knocked out bydisrupting the gene with a kanamycin-resistance cassette (Maio and Lam(1995) Plant J. 7:359-365). The targeting cassette had about 4 kb ofhomology to the 5′ end of TGA3 and about 3 kb of homology to the 3′ endof the gene. In another report, the AGL5 MADS-box gene has been knockedout by homologous recombination in Arabidopsis (Kempin et al. 1997Nature 389:802-803). The targeting construct consisted of akanamycin-resistance cassette inserted into the AGL5 sequence roughly 3kb from the 5′ end and 2 kb from the 3′ end.

[0053] In animals, more specific categories of target sequences includegenes involved in various diseases or medical conditions, such ascancer, including targets such as tumor suppressor genes (e.g., p53,p73, p51, or p40) and oncogenes (e.g., c-myc, c-jn, c-fos, c-rel, c-qin,c-neu, c-src, c-abl, c-lck, c-mil/raf, c-ras, c-sis, or c-fps); obesity,fertility, diabetes, hypertension, coronary disease, neurologicaldisorders, cystic fibrosis, multiple sclerosis, muscular dystrophy,genetic disorders, and the like.

[0054] Target Modifying Sequences, Homologous Recombination, and GeneTargeting

[0055] Homologous recombination is recombination occurring as a resultof interaction between segments of genetic material that is homologousover a sufficient length of nucleotide sequence. Homologousrecombination is an enzyme-catalyzed process that occurs in essentiallyall cell types. The reaction takes place when nucleotide strands ofhomologous sequence are aligned in proximity to one another, and entailsbreaking phosphodiester bonds in the nucleotide strands and rejoiningwith neighboring homologous strands or with an homologous sequence onthe same strand. The breaking and rejoining can occur with precision,such that the sequence fidelity is retained.

[0056] The frequency of homologous recombination is influenced by anumber of factors. Different organisms vary with respect to the amountof homologous recombination that occurs in their cells and the relativeproportion of homologous to non-homologous recombination that occurs isalso species-variable. Generally, the length of the region of homologyaffects the frequency of homologous recombination events, the longer theregion of homology, the greater the frequency. The length of thehomology region needed to observe homologous recombination is alsospecies-variable. In many cases, at least 5 kb of homology has beenutilized, but homologous recombination has been observed with as littleas 25-50 bp of homology. The minimum length of homology needed has beenestimated at 20-50 bp in E. coli (Singeret al. (1982) Cell 31:25-33;Shen & Huang (1986) Genetics 112:441-457; Watt et al. (1985) PNAS82:4768-4772), 63-89 bp in S. cerevisaie (Sugawara & Haber (1992) Mol.Cell. Biol. 12:563-575), and 163-300 bp in mammalian cells (Rubnitz &Subramani (1984) Mol. Cell. Biol. 4:2253-2258; Ayares et al. (1986) PNAS83:5199-5203; Liskay et al. (1987) Genetics 115:161-167).

[0057] However, differences in the frequency of homologous recombinationcan be offset somewhat by sensitive selection for recombinations that dooccur. Other factors, such as the degree of homology between the donor(target modifying polynucleotide) and target sequence will alsoinfluence the frequency of homologous recombination events, as iswell-understood in the art. In ES cells, Te Riele et al. observed thatuse of targeting constructs based on isogenic DNA resulted in a 20-foldincrease in targeting efficiency (Te Riele et al. (1992) PNAS89:5128-5132). They concluded that base sequence divergence betweennon-isogenic DNA sources was the major influence on homologousrecombination efficiency. Absolute limits for the length of homology orthe degree of homology cannot be fixed, but depend on the number ofevents that can be generated, screened, and selected. All such factorsare well known in the art, and can be taken into account when using theinvention for gene targeting in any given organism.

[0058] Gene targeting has been demonstrated in insects. In Drosophila,Dray and Gloor found that as little as 3 kb of total template:targethomology sufficed to copy a large non-homologous segment of DNA into thetarget with reasonable efficiency. (Genetics 147:689-699 1997). UsingFLP-mediated DNA integration at a target FRT in Drosophila, Golic (Golicet al. (1997) Nucl. Acids Res. 25:3665) showed integration wasapproximately 10-fold more efficient when the donor and target shared4.1 kb of homology compared to 1.1 kb of homology. Therefore, data fromDrosophila indicates that 2-4 kb of homology is sufficient for efficienttargeting, but there is some evidence that much less homology maysuffice, on the order of about 30 bp to about 100 bp (Nassif & Engels(1993) PNAS 90:1262-1266; Keeler & Gloor (1997) Mol. Cell Biol.17:627-634).

[0059] Gene targeting has been demonstrated in plants. The parametersfor gene targeting in plants have primarily been investigated byrescuing introduced truncated selectable marker genes. In theseexperiments, the homologous DNA fragments for homologous recombinationwere typically between 0.3 kb to 2 kb. Observed frequencies forhomologous recombination were on the order of 10⁻⁴-10⁻⁵. See, forexample, Halfter et al. (1992) Mol. Gen. Genet. 231:186-193; Offringa etal. (1990) EMBO 9:3077-3084; Offringa et al. (1993) PNAS 90:7346-7350;Paszkowski et al. (1988) EMBO 7:4021-4026; Hourda and Paszkowski (1994)Mol. Gen. Genet. 243:106-111; and Risseeuw et al. (1995) Plant J.7:109-119.

[0060] An endogenous, non-selectable gene was targeted in Arabidopsis.The targeting vector contained a region of about 7 kb homologous to thetarget gene and the targeting frequency was estimated to be at least3.9×10⁻⁴ (Maio and Lam (1995) Plant J. 7:359-365).

[0061] Using a positive-negative selection scheme and a targeting vectorcontaining up to 22.9 kb of sequence homologous to the target, Thykjaerand co-workers detected gene targeting with a frequency less than5.3×10⁻⁵, despite the large flanking sequences available forrecombination (Thykjaer et al. (1997) Plant Mol. Biol. 35:523-530). InArabidopsis, the AGL5 MADS-box gene was knocked out by homologousrecombination (Kempin et al. (1997) Nature 389:802-803) using atargeting construct consisting of a kanamycin-resistance cassetteinserted into the AGL5 sequence roughly 3 kb from the 5′ end and 2 kbfrom the 3′ end. Of the 750 kanamycin-resistant transgenic lines thatwere generated, one line contained the anticipated insertion.

[0062] Gene targeting has also been accomplished in other organisms. Forexample, at least 150-200 bp of homology was required for homologousrecombination in the parasitic protozoan Leishmania, regions less than 1kb a decrease in the length had a linear effect on the targetingfrequency, and the targeting frequency plateaus at 1-2 kb of homology(Papadopoulou and Dumas (1997) Nucl. Acids Res. 25:4278-4286). In thefilamentous fungus Aspergillus nidulans, gene replacement has beenaccomplished with as little as 50 bp flanking homology (Chaveroche etal. (2000) Nucl. Acids Res. 28(22):e97). Targeted gene replacement hasalso been demonstrated in the ciliate Tetrahymena thermophila (Gaertiget al. (1994) Nucl. Acids Res. 22:5391-5398).

[0063] In mammals, gene targeting has been most successful in the mouseas pluripotent embryonic stem cell lines exists (ES) that can be grownin culture, transformed, selected and introduced into an embryonic stageof a mouse embryo. Embryos bearing inserted transgenic ES cells developas genetically chimeric offspring. By interbreeding siblings, homozygousmice carrying the selected genes can be obtained. An overview of theprocess is provided in Watson et al. (1992) Recombinant DNA, 2^(nd) Ed.,Scientific American Books distributed by W H Freeman & Co.; Capecchi, MR (1989) Trends in Genetics 5:70-76; and Bronson, SK (1994) J. Biol.Chem. 269:27155-27158. Both homologous and non-homologous recombinationoccur in mammalian cells. While both processes occur with low frequency,non-homologous recombination occurs more frequently than homologousrecombination. ES cells are transfected with a DNA construct thatcombines a donor DNA having the modification to be introduced at thetarget site combined with flanking sequence homologous to the targetsite, and marker genes as needed for selection, as well as any otherdesired sequences. The donor construct need not be integrated into thegenome initially, but can recombine with the target site by homologousrecombination, or become integrated by non-homologous recombination.Since homologous recombination events are rare, dual selection can beused to select for gene targeted events and to select against randomintegration. The selections are conveniently carried out in vitro on EScells in culture. Other screening, such as PCR, can also be used toidentify desired events. In general, the frequency of homologousrecombination is increased as the length of the region of homology inthe donor is increased, with at least 5 kb of homology commonly used.However, homologous recombination has been observed with as little as25-50 bp of homology. It has been observed that small deletions orinsertions into the target site are introduced with higher frequencythan point mutations, but any desired modification can be obtained byappropriate design of donor vector, and selection and/or screeningmethods.

[0064] In an effort to create a mouse model system for cystic fibrosisKoller et al. used a targeting construct to disrupt exon 10 of the CTFRgene (Koller et al. (1991) PNAS 88:10730-10734). The construct sharedhomology to 7.8 kb of the target, spanning exon 10, and replaced part ofthe exon with two neo genes which causes a premature stop codon. A genetargeting frequency of 4×10⁻⁴ was observed in ES cells.

[0065] In ES cells comprising two renin genes (Ren-1D and Ren-2) whichshare about 95% sequence identity at the genomic level, a targetingconstruct with about 5.5 kb of homology across exons 2-5 of Ren-1Dspecifically recombined only with the target gene with a gene targetingfrequency of 5.29×10⁻³ (Miller et al. (1992) PNAS 89:5020-5024). It wasestimated that the targeting frequency observed was enhanced about2.7-fold by the inclusion of a negative selectable marker in thetargeting construct.

[0066] In order to study the transcriptional control of type I collagen,the first intron of ColIA1 was targeted in mouse ES cells (Hormuzdi etal. (1998) Mol. Cell. Biol. 18:3368-3375). The targeting construct,which shared about 13 kb of homology to the target, resulted in a 1.3 kbdeletion in intron 1. Even though there is a large deletion in the firstintron, the study showed the intron was still correctly spliced.

[0067] A point mutation in β-globin causes sickle cell disease. Using amouse-human hybrid cell line, BSM, which contains human chromosome 11,the sickle cell allele β^(S)-globin was corrected to the normalβ^(A)-globin allele (Shesley et al. (1991) PNAS 88:42944298). Thetargeting vector comprised 4.7 kb of homology to the β-globin gene, aswell as a selectable marker outside of the target gene, and resulted ina gene targeting frequency of at least 1×10⁴.

[0068] Gene targeting in mammals other than mouse has been limited bythe lack of stem cells capable of being transplanted to oocytes ordeveloping embryos. However, McCreath, K J et al. (2000) Nature405:1066-1069 have reported successful gene targeting in sheep bytransformation and selection in primary embryo fibroblast cells. Thetargeted fibroblast nuclei were transferred to enucleated egg cellsfollowed by implantation in the uterus of a host mother. The techniqueyields a homozygous, non-chimeric offspring, however the time availablefor targeting and selection is short.

[0069] The organisms which can be used in the invention include, but arenot limited to: insects, including Coleoptera, Diptera, such asDrosophila, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, andOrthoptera; plants, including both monocotyledonous and dicotyledonousplants such as, but not limited to, maize, rice, wheat, oats, barley,sorghum, millet, soybean and other legumes, canola, Brassica, alfalfa,sunflower, safflower, Arabidopsis, cotton, potato, tomato, tobacco andthe like; animals including mice, rats, sheep, pigs, bovines,amphibians, such as Xenopus, fish, such as zebrafish, birds;invertebrates such as C. elegans; fungi (Chaveroche et al. (2000) Nucl.Acids Res. 28(22):e97; DeLozane and Spudich (1987) Science236:1086-1091); and protozoa such as ciliates (Gaertig et al. (1994)Nucl. Acids Res. 22:5891-5398), and/or parasitic protozoa (Papadopoulouand Dumas (1997) Nucl. Acids Res. 25:4278-4286), and the like.

[0070] The targeted event can be effected in the whole organism, orlimited to certain tissue or cell types or even particular subcellularorganelles. For example, homologous recombination has been used totarget foreign genes into the plastid genome in tobacco (Zoubenko et al.(1994) Nucl. Acids Res. 22:3819-3824), and to correct a defective genein hematopoietic progenitor cells (Hatada et al. (2000) PNAS97:13807-13811).

[0071] The amount of homology shared between the target and the targetmodifying polynucleotide can vary and includes unit integral values inthe ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp,150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp,450-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10kb, or up to and including the total length of the target site. Theseranges include every integer within the range, for example, the range of1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, and 20 bp.

[0072] Nucleic Acids

[0073] Polynucleotides, including targeting vectors, target modifyingpolynucleotides, replicase polynucleotides, origins of replication,recombinase polynucleotides, transposon polynucleotides, transposasepolynucleotides, selectable markers, and any other polynucleotides ofinterest, useful in the present invention can be obtained using (a)standard recombinant methods, (b) synthetic techniques, or combinationsthereof. In general, examples of appropriate molecular biologicaltechniques and instructions are found in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory,Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to MolecularCloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press,Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et a/.,Eds., Greene Publishing and Wiley-Interscience, New York (1995); PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997), all are herein incorporated by reference.

[0074] Polynucleotides and functional variants useful in the inventioncan be obtained using primers that selectively hybridize under stringentconditions. Primers are generally at least 12 bases in length and can beas high as 200 bases, but will generally be from 15 to 75, typicallyfrom 15 to 50. Functional fragments can be identified using a variety oftechniques such as restriction analysis, Southern analysis, primerextension analysis, and DNA sequence analysis.

[0075] Variants of the nucleic acids can be obtained, for example, byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like. See, forexample, Ausubel, pages 8.0.3-8.5.9. Also, see generally, McPherson(ed.), DIRECTED MUTAGENESIS: A Practical approach, (IRL Press, 1991).Thus, the present invention also encompasses DNA molecules comprisingnucleotide sequences that have substantial sequence similarity with theinventive sequences. Conservatively modified variants are preferred.

[0076] Nucleic acids produced by sequence shuffling of usefulpolynucleotides can also be used. Sequence shuffling is described in PCTpublication No. 96/19256. See also, Zhang, J.-H., et al. Proc. Natl.Acad. Sci. USA 94:4504-4509 (1997).

[0077] Also useful are 5′ and/or 3′ UTR regions for modulation oftranslation of heterologous coding sequences. Positive sequence motifsinclude translational initiation consensus sequences (Kozak, NucleicAcids Res. 15:8125 (1987)) and the 7-methylguanosine cap structure(Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elementsinclude stable intramolecular 5′ UTR stem-loop structures (Muesing etal., Cell 48:691 (1987)) and AUG sequences or short reading frames 5′ ofthe appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al., Mol. andCell. Biol. 8:284 (1988)).

[0078] Further, the polypeptide-encoding segments of the polynucleotidescan be modified to alter codon usage. Codon usage in the coding regionsof the polynucleotides of the present invention can be analyzedstatistically using commercially available software packages such as“Codon Preference” available from the University of Wisconsin GeneticsComputer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395(1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).

[0079] For example, the polynucleotides can be optimized for enhanced orsuppressed expression in the target organism. In the case of plants,see, for example, EPA0359472; WO91/16432; Perlak et al. (1991) Proc.Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) NucleicAcids Res. 17:477-498, the disclosures of which are incorporated hereinby reference. In this manner, the genes can be synthesized utilizingspecies-preferred codons.

[0080] The nucleic acids may conveniently comprise a multi-cloning sitecomprising one or more endonuclease restriction sites inserted into thenucleic acid to aid in isolation of the polynucleotide. Also,translatable sequences may be inserted to aid in the isolation of thetranslated polynucleotide of the present invention. For example, ahexa-histidine marker sequence provides a convenient means to purify theproteins of the present invention.

[0081] The polynucleotides can be attached to a vector, adapter,promoter, transit peptide or linker for cloning and/or expression of apolynucleotide of the present invention. Additional sequences may beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide, or to improve the introduction of the polynucleotideinto a cell. Use of cloning vectors, expression vectors, adapters, andlinkers is well known and extensively described in the art. For adescription of such nucleic acids see, for example, Stratagene CloningSystems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, AmershamLife Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

[0082] The targeting vectors may comprise large regions of DNA withhomology to the target site. Examples of construction of targetingvectors with large fragments of DNA are found in Lalioti and Heath(2001) Nucl. Acids Res. 29(3):e14; Akiyama et al. (2000) Nucl. AcidsRes. 28(16):e77; and Angrand et al. (1999) Nucl. Acids Res. 27(17):e16.Transformation-associated recombination (TAR) cloning methods may alsobe used to isolate large regions of DNA. Examples of minimal homologyrequired and selection of clones are found in Noskov et a/. (2001) Nucl.Acids Res. 29(6):e32 and Noskov et al. (2002) Nucl. Acids Res. 30(2):e8.

[0083] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol.152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

[0084] The genomic library can be screened using a probe based upon thesequence of a nucleic acid used in the present invention. Those of skillin the art will appreciate that various degrees of stringency ofhybridization can be employed in the assay; and either the hybridizationor the wash medium can be stringent. The degree of stringency can becontrolled by temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide.

[0085] Typically, stringent hybridization conditions will be those inwhich the salt concentration is less than about 1.5 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

[0086] The hybridization can be conducted under low stringencyconditions which include hybridization with a buffer solution of 30%formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and awash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50°C. In another option, the hybridization can be conducted under moderatestringency conditions which include hybridization in 40% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° C. In yetanother option, the hybridization can be conducted under high stringencyconditions which include hybridization in 50% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.1×SSC at 60° C.

[0087] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

[0088] The nucleic acids can be amplified from nucleic acid samplesusing amplification techniques. For instance, polymerase chain reaction(PCR) technology can be used to amplify the sequences of polynucleotidesof the present invention and related genes directly from genomic DNA orlibraries. PCR and other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes.

[0089] Examples of techniques useful for in vitro amplification methodsare found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methodsand Applications, Innis et al., Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used toimprove yield of long PCR products.

[0090] PCR-based screening methods have also been described. Wilfingeret al. describe a PCR-based method in which the longest cDNA isidentified in the first step so that incomplete clones can be eliminatedfrom study. BioTechniques, 22(3):481-486 (1997).

[0091] The nucleic acids can also be prepared by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett 22:1859-1862 (1981); the solidphase phosphoramidite triester method described by Beaucage andCaruthers, Tetra. Letts. 22(20):1859-1862 (1981), e.g., using anautomated synthesizer, e.g., as described in Needham-VanDevanter et al.,Nucleic Acids Res., 12:6159-6168 (1984); and, the solid support methodof U.S. Pat. No. 4,458,066.

[0092] Expression cassettes comprising the isolated polynucleotidesequences of interest are also provided. An expression cassette willtypically comprise a polynucleotide operably linked to transcriptionalinitiation regulatory sequences that will direct the transcription ofthe polynucleotide in the intended host cell, such as tissues of atransformed plant.

[0093] The construction of expression cassettes that can be employed inconjunction with the present invention is well known to those of skillin the art in light of the present disclosure. See, e.g., Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor,N.Y.; Gelvin et al. (1990) Plant Molecular Biology Manual; Prakash etal. eds. (1993) Plant Biotechnology: Commercial Prospects and Problems,Oxford & IBH Publishing Co., New Delhi, India; and Heslot et al. (1992)Molecular Biology and Genetic Engineering of Yeasts CRC Press, Inc.,USA; each incorporated herein in its entirety by reference.

[0094] For example, expression cassettes may include (1) a nucleic acidunder the transcriptional control of 5′ and 3′ regulatory sequences and(2) a dominant selectable marker. Such plant expression cassettes mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible, constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0095] Constitutive, tissue-preferred or inducible promoters can beemployed. Examples of constitutive promoters include the cauliflowermosaic virus (CaMV) 35S transcription initiation region, the 1′- or2′-promoter derived from T-DNA of Agrobacterium tumefaciens, theubiquitin 1 promoter, the Smas promoter, the cinnamyl alcoholdehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, thepEmu promoter, the rubisco promoter, the GRP1-8 promoter and othertranscription initiation regions from various plant genes known to thoseof skill.

[0096] Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, the PPDK promoter and the pepcarboxylasepromoter which are both inducible by light. Also useful are promoterswhich are chemically inducible, such as the In2-2 promoter which issafener induced (U.S. Pat. No. 5,364,780), the ERE promoter which isestrogen induced, and the Axig1 promoter which is auxin induced andtapetum specific but also active in callus (PCT US01/22169).

[0097] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruit, seeds, or flowers. An exemplary promoteris the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051). Examples of seed-preferred promoters include, but are notlimited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A. etal. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res.18(21):6426; and Kloesgen, R. B. et al. (1986) Mol. Gen. Genet.203:237-244. Promoters that express in the embryo, pericarp, andendosperm are disclosed in U.S. Pat. No. 6,225,529 and PCT publicationWO 00/12733. The disclosures each of these are incorporated herein byreference in their entirety.

[0098] Either heterologous or non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue.

[0099] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or even from any other eukaryotic gene.

[0100] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).

[0101] The vector comprising the polynucleotide sequences useful in thepresent invention will typically comprise a marker gene that confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic or herbicide resistance. Suitable genes includethose coding for resistance to the antibiotic spectinomycin orstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance.

[0102] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to inhibitaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron.

[0103] Typical vectors useful for expression of nucleic acids in higherplants are well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol., 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl.Acad. Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.). A variety of plant viruses that can be employed asvectors are known in the art and include cauliflower mosaic virus(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0104] Useful polynucleotides can be expressed in either sense oranti-sense orientation as desired. In plant cells, it has been shownthat antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat'l. Acad. Sci. (USA) 85: 8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

[0105] Another method of suppression is sense suppression. For anexample of the use of this method to modulate expression of endogenousgenes see, Napoli et al., The Plant Cell 2: 279-289 (1990) and U.S. Pat.No. 5,034,323. Another method of down-regulation of the protein involvesusing PEST sequences that provide a target for degradation of theprotein.

[0106] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591 (1988).

[0107] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B.,et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinkingto a target nucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203.Use of crosslinking in triple-helix forming probes was also disclosed byHome et al., J Am Chem Soc (1990) 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-strandedoligonucleotides has also been described by Webb and Matteucci, J AmChem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674;Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds tobind, detect, label, and/or cleave nucleic acids are known in the art.See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;5,256,648; and, 5,681,941.

[0108] Proteins useful in the present invention include proteins derivedfrom the native protein by deletion (so-called truncation), addition orsubstitution of one or more amino acids at one or more sites in thenative protein. In constructing variants of the proteins of interest,modifications will be made such that variants continue to possess thedesired activity.

[0109] For example, amino acid sequence variants of the polypeptide canbe prepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

[0110] The present invention includes catalytically active polypeptides(i.e., enzymes). Catalytically active polypeptides will generally have aspecific activity of at least 20%, 30%, or 40%, or at least 50%, 60%, or70%, or at least 80%, 90%, 95%, or 100% that of the native(non-synthetic), endogenous polypeptide. Further, the substratespecificity (k_(cat)/K_(m)) is optionally substantially similar to thenative (non-synthetic), endogenous polypeptide. Typically, the Km willbe at least 30%, 40%, or 50%, that of the native (non-synthetic),endogenous polypeptide; or at least 60%, 70%, 80%, 90%, 95% or 100%.Methods of assaying and quantifying measures of enzymatic activity andsubstrate specificity (k_(cat)/K_(m)), are well known to those of skillin the art.

[0111] The methods of the present invention can be used with any cellsuch as bacteria, yeast, insect, mammalian, or plant cells. Thetransformed cells produce viral replicase protein.

[0112] An intermediate host cell may be used in the practice of thisinvention to increase the copy number of the targeting vector, and/orreplicase, recombinase, or transposase expression cassettes. With anincreased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired target host cells. Intermediate host cells that can be usedin the practice of this invention include prokaryotes, includingbacterial hosts such as Eschericia coli, Salmonella typhimurium, andSerratia marcescens. Eukaryotic hosts such as yeast or filamentous fungimay also be used in this invention. One can use target host specificpromoters that do not cause expression of the polypeptide in bacteria.

[0113] Commonly used prokaryotic control sequences include promoterssuch as the beta lactamase (penicillinase) and lactose (lac) promotersystems (Chang et al., Nature 198:1056 (1977)), the tryptophan (trp)promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980)) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatakeet al., Nature 292:128 (1981)). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracycline,or chloramphenicol.

[0114] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22: 229-235; Mosbach et al. (1983) Nature 302: 543-545).

[0115] Synthesis of heterologous proteins in yeast is well known. SeeSherman, F. et al. Methods in Yeast Genetics, Cold Spring HarborLaboratory (1982). Two widely utilized yeast for production ofeukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

[0116] The protein can be isolated from yeast by lysing the cells andapplying standard protein isolation techniques to the lysates. Themonitoring of the purification process can be accomplished by usingWestern blot techniques or radioimmunoassay of other standardimmunoassay techniques.

[0117] The proteins useful in the present invention can also beconstructed using non-cellular synthetic methods. Techniques for solidphase synthesis are described by Barany and Merrifield, Solid-PhasePeptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.;Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewartetal., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford,III. (1984). Proteins of greater length may be synthesized bycondensation of the amino and carboxy termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxy terminal end(e.g., by the use of the coupling reagent N,N′-dicyclohexylcarbodiimide)are known to those of skill.

[0118] The proteins useful in this invention may be purified tosubstantial purity by standard techniques well known in the art,including detergent solubilization, selective precipitation with suchsubstances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. Detection of the expressed proteinis achieved by methods known in the art, for example, radioimmunoassays,Western blotting techniques or immunoprecipitation.

[0119] In certain embodiments, the invention can be practiced in a widerange of plants such as monocots and dicots. For example, the methods ofthe present invention can be employed in corn, soybean, sunflower,safflower, potato, tomato, sorghum, canola, wheat, alfalfa, cotton,rice, barley and millet.

[0120] Transformation

[0121] The method of transformation/transfection is not critical to theinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform hostcells they may be directly applied. Accordingly, a wide variety ofmethods have been developed to insert a DNA sequence into the genome ofa host cell to obtain the transcription and/or translation of thesequence to effect phenotypic changes in the organism. Thus, any methodthat provides for efficient transformation/transfection may be employed.

[0122] A DNA sequence coding for the desired polynucleotide useful inthe present invention, for example a cDNA, RNA or a genomic sequence,will be used to construct an expression cassette that can be introducedinto the desired host. Isolated nucleic acid acids of the presentinvention can be introduced according techniques known in the art.Generally, expression cassettes as described above and suitable fortransformation of are prepared.

[0123] For single-celled organisms and organisms that can be regeneratedfrom single cells, transformation can be carried out by in vitroculture, followed by selection for transformation and regeneration oftransformants. Methods often used for transferring DNA or RNA into cellsinclude microinjection, particle gun bombardment, forming DNA or RNAcomplexes with cationic lipids, liposomes or other carrier materials,electroporation, chemical methods, and viral methods. Other techniquesare known in the art, for example see standard reference works such asMethods in Enzymology, Methods in Cell Biology, Molecular BiologyTechniques, all published by Academic Press, Inc. NY. Methods fortransforming various host cells are disclosed in Klein et al.“Transformation of microbes, plants and animals by particlebombardment”, Bio/Technol. New York, N.Y., Nature Publishing Company,March 1992, 10(3):286-291. Waters has recently demonstrated the stabletransfer of nucleic acids from bacteria to cultured mammalian cells,apparently via cell conjugation (Waters, VL 2001 Nature Genetics29:375-376).

[0124] Transfer of the polynucleotide into the cell nucleus occurs bycellular processes and can sometimes be aided by choice of anappropriate vector, by including integration site sequences which can beacted upon by an intracellular transposase or recombinase. For reviewsof transposase or recombinase mediated integration see, e.g., Craig, NLK(1988) Ann Rev Genet 22:77; Cox, MM (1988) In Genetic Recombination(Kucherlapati and Smith, Eds.) pp. 429-443, American Society forMicrobiology, Washington, D.C.; Hoess, RH et al. (1990) In Nucleic Acidand Molecular Biology (Eckstein and Lilley, Eds.) Vol 4, pp. 99-109,Springer-Verlag, Berlin.

[0125] Direct transformation of multicellular organisms can often beaccomplished at an embryonic stage of the organism. For example, inDrosophila, as well as other insects, DNA can be microinjected into theembryo at a multinucleate stage where it can become integrated into manynuclei, some of which become the nuclei of germ line cells. Recently,stable germlne transformations were reported in mosquito (Catteruccia,F., et al. (2000) Nature 405:954-962). By incorporating a marker as acomponent of the transforming DNA, non-chimeric progeny of the originaltransformant can be identified and maintained. Direct microinjectioninto egg or embryo cells has also been employed effectively fortransforming many species, Xenopus for example. In the mouse, theexistence of pluripotent embryonic stem (ES) cells that are amenable toculture in vitro has been used to generate transformed mice. The EScells can be transformed in culture, then microinjected into mouseblastocysts, where they integrate into the developing embryo andgenerate germline chimeras. By interbreeding heterozygous siblings,homozygous animals carrying the desired gene can be obtained. Forreviews of the methods for transforming multicellular organisms see,e.g. Haren et al. (1999) Ann Rev Microbiol 53:245-281; Reznikoff et al.(1999) Biochem Biophys Res Comm 266(3):729-734; Ivics et al. (1999)Methods in Cell Biology 60:99-131; Weinberg, E S (1998) Curr Biol8(7):R244-247; Hall et al. (1997) FEMS Microbiol Rev 21(2):157-178;Craig (1997) Ann Rev Biochem 66:437-474; Beall et al. (1997) Genes Dev11(16):2137-2151.

[0126] Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical, scientific, andpatent literature. See, for example, Weising et al., Ann. Rev. Genet 22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG-mediated transfection, particle bombardment,silicon fiber delivery, or microinjection of plant cell protoplasts orembryogenic callus. See, e.g., Tomes, et al., Direct DNA Transfer intoIntact Plant Cells Via Microprojectile Bombardment. pp.197-213 in PlantCell, Tissue and Organ Culture, Fundamental Methods. eds. O. L. Gamborgand G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995.Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0127] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3: 2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327:70-73 (1987).

[0128]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233: 496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80: 4803 (1983). For instance, Agrobacterium transformation ofmaize is described in U.S. Pat. No. 5,550,318.

[0129] Other methods of transformation include (1) Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, vol. 6, P W J Rigby, Ed., London, AcademicPress, 1987; and Lichtenstein, C. P., and Draper, J. In: DNA Cloning,Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use ofA. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353,1984), (3) the vortexingmethod (see, e.g., Kindle, Proc. Natl. Acad. Sci., USA 87:1228, (1990).

[0130] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingnucleic acids can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena et al., Nature, 325:274 (1987).DNA can also be injected directly into the cells of immature embryos andthe rehydration of desiccated embryos as described by Neuhaus et al.,Theor. Appl. Genet., 75:30 (1987); and Benbrook et al., in ProceedingsBio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).

[0131] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

[0132] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell, 2:603-618 (1990).

[0133] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp.124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

[0134] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.,80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0135] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). For maize cell culture andregeneration see generally, The Maize Handbook, Freeling and Walbot,Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition,Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis.(1988).

[0136] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

[0137] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated polynucleotide of interest. Progeny and variants, and mutantsof the regenerated plants are also included within the scope of theinvention, provided that these parts comprise the introduced nucleicacid sequences.

[0138] Transgenic plants expressing a selectable marker can be screenedfor transmission of the polynucleotide of interest, for example,standard DNA detection techniques to detect the polynucleotide, and/orimmunoblots to detect protein expression. Transgenic lines are alsotypically evaluated on levels of expression of the heterologous nucleicacid. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can then analyzedfor protein expression by Western immunoblot analysis using thespecifically reactive antibodies of the present invention. In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0139] Plants that can be used in the method of the invention varybroadly and include monocotyledonous and dicotyledonous plants includingcorn, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley, potato, tomato, and millet.

[0140] Seeds derived from plants regenerated from transformed plantcells, plant parts or plant tissues, or progeny derived from theregenerated transformed plants, may be used directly as feed or food, orfurther processing may occur.

[0141] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed. Further, one of skill will recognize that anycomponent of the method can be introduced to the host organism bysexually crossing the target host with a donor organism which comprisesone of more of the following: a targeting vector, and/or a replicaseexpression cassette, a site-specific recombinase expression cassette, ora transposase expression cassette. In the case of cells in culture, thecomponents may also be brought together by fusing target cells and donorcells.

[0142] Any method described above in reference to plants can be appliedto the generation and identification of gene targeting events in othertarget host organisms. Examples of organisms which can be used in theinvention include, but are not limited to: insects, includingColeoptera, Diptera, such as Drosophila, Hemiptera, Homoptera,Hymenoptera, Lepidoptera, and Orthoptera; plants, including bothmonocotyledonous and dicotyledonous plants such as, but not limited to,maize, rice, wheat, oats, barley, sorghum, millet, soybean and otherlegumes, canola, Brassica, alfalfa, sunflower, safflower, Arabidopsis,cotton, potato, tomato, tobacco and the like; animals including mice,rats, sheep, pigs, bovines, amphibians, such as Xenopus, fish, such aszebrafish, birds; and protozoa such as ciliates, and/or parasiticprotozoa, and the like.

[0143] Identification and Characterization of Modified Target Cells andOrganisms

[0144] Gene targeting can be performed without selection, if there is asensitive method for identifying recombinants, for example if thetargeted gene modification can be easily detected by PCR analysis, or ifit results in a certain phenotype. However, in most cases,identification of gene targeting events will be facilitated by the useof markers. Markers useful in the invention include positive andnegative selectable markers as well as markers that facilitatescreening, such as visual markers. Selectable markers include genescarrying resistance to an antibiotic such as spectinomycin (e.g. theaada gene), streptomycin (e.g., aada, or SPT), kanamycin (e.g., nptll),hygromycin (e.g., HPT), gentamycin, phleomycin, zeocin, or bleomycin, orresistance to a herbicide such as phosphinothricin (bar gene), orsulfonylurea (acetolactate synthase—ALS), genes that fulfill a growthrequirement on an incomplete media such as HIS3, LEU2, URA3, LYS2, andTRP1 genes in yeast, and other such genes known in the art. Negativeselectable markers include cytosine deaminase (codA) (Stougaard 1993Plant J. 3:755-761), tms2 (DePicker et al. 1988 Plant Cell Rep.7:63-66), nitrate reductase (Nussame et al. 1991 Plant J. 1:267-274),and SU1 (O'Keefe et al. 1994 Plant Physiol. 105:473-482). Screenablemarkers include fluorescent proteins such as green fluorescent protein(GFP) (Chalfie et al., 1994 Science 263:802; U.S. Pat. No. 6,146,826;U.S. Pat. No. 5,491,084; and WO 97/41228), reporter enzymes such asβ-glucuronidase (GUS) (Jefferson R. A. 1987 Plant Mol. Biol. Rep. 5:387;U.S. Pat. No. 5,599,670; and U.S. Pat. No. 5,432,081), β-galactosidase(lacZ), alkaline phosphatase (AP), glutathione S-transferase (GST) andluciferase (U.S. Pat. No. 5,674,713; and Ow et al. 1986 Science234(4778):856-859), visual markers such as color markers likeanthocyanins such as CRC (Ludwig et al. 1990 Science 247(4841):449-450)R gene family (e.g. Lc, P, S), A, C, R-nj, body and/or eye color genesin Drosophila, and coat color genes in mammalian systems, and othersknown in the art.

[0145] One or more markers may be used in order to select and screen forgene targeting events. One common strategy for gene disruption involvesusing a target modifying polynucleotide in which the target is disruptedby a promoterless selectable marker. Since the selectable marker lacks apromoter, random integration events are unlikely to lead totranscription of the gene. Gene targeting events will put the selectablemarker under control of the promoter for the target gene. Gene targetingevents are identified by selection for expression of the selectablemarker. Another common strategy utilizes a positive-negative selectionscheme. This scheme utilizes two selectable markers, one that confersresistance (R⁺) coupled with one that confers a sensitivity (S⁺), eachwith a promoter. When this polynucleotide is randomly inserted, theresulting phenotype is R⁺/S⁺. When a gene targeting event is generated,the two markers are uncoupled and the resulting phenotype is R⁺/S⁻.Examples of using positive-negative selection are found in Thykjaer etal. (1997) Plant Mol Biol. 35:523-530; and WO 01/66717, which are hereinincorporated by reference.

[0146] Cells or organisms identified by one or more selective markerscan be further screened for modification of the target polynucleotide ofinterest by a large number of molecular and biochemical assays known inthe art. For example, standard DNA detection techniques to detect thepolynucleotide including amplification techniques such as restrictionenzyme analysis, PCR, Southern and Northern blots, DNA chips, in situhybridization, sequencing and the like. PCR is fast, specific andsensitive method commonly used to detect gene targeting events. Primersthat distinguish between unmodified and modified target are designed andamplification conditions identified as known to those of skill in theart, see for example standard references such as Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual 2^(nd) Ed. Cold Spring HarborPress New York, Walker and Gaastra, eds. (1983) Techniques in MolecularBiology, MacMillan Publishing New York, Innis et al. eds. (1990) PCRProtocols: A Guide to Methods and Applications, Academic Press, Inc. SanDiego, Calif., Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, New York.See also, Kim and Smithies (1988) Nucl. Acids Res. 16:8887-8903.Biochemical and/or immunochemical assay to detect and/or quantifyprotein expression may also be employed, such as immunoblots,immunoprecipitation, ELISA assays, immunohistochemistry, enzyme activityassays, enzyme kinetic studies, chromatographic and electrophoreticseparations such as polyacrylamide gel profiles, capillaryelectrophoresis, protein binding/interaction assays such as ligandbinding, gel shift assays, blot overlays, co-immunoprecipitation, andthe like. Standard techniques for RNA analysis can be employed andinclude PCR amplification assays using oligonucleotide primers designedto amplify only the modified RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. In addition, insitu hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue.

[0147] The present invention will be further described by reference tothe following detailed examples.

[0148] It is understood, however, that there are many extensions,variations, and modifications on the basic theme of the presentinvention beyond that shown in the examples and description, which arewithin the spirit and scope of the present invention. All publications,patents, and patent applications cited herein are hereby incorporated byreference.

EXAMPLES Example 1 Replicating Vectors and Recombination

[0149] Vector construction was done using standard molecular biologytechniques. T-DNA vectors were constructed to test whether recombinantT-DNA molecules could be produced and could persist in transformed maizecells using a viral replication mechanism acting in concert withrecombination. The recombination event could be site-specific,homologous or illegitimate recombination. The basic vector designcomprised at least one source of microhomology that could be used tocircularize the T-DNA. For example, overlapping areas of the neo gene,FRTI sites, or the left and right T-DNA borders can be used as theregions of microhomology. A recombination event within the neo gene,FRT1 sites, or the border sequences should generate replicationcompetent, circular T-DNAs which therefore leads to the activation of arecombination marker gene, for example neo which conferskanamycin-resistance, or gusA. In order to recover replicating T-DNAmolecules from E. coli, the ampicillin-resistance gene was incorporatedinto the T-DNA structure. A FLP recombinase gene, driven by a separatepromoter, can be provided on the same T-DNA or provided by anothervector.

[0150] A. The Effect of Replicase Expression on Homologous Recombinationof T-DNA TABLE 1 Plasmid Description Neo Rep P10525RB-Ubipro/intron-GUS-Ubi-Bar-LB No No P16821LB-3′Δneo-Rep-WDVLIR-Ubipro/intron- Inactive Yes GUS-AMP-5′Δneo-RBP16822 RB-3′Δneo-Rep-WDVLIR-Ubipro/intron- Inactive YesGUS-AMP-5′Δneo-LB P16823 RB-3′Δneo-WDVLIR-Ubipro/intron- Inactive NoGUS-AMP-5′Δneo-LB P16824 RB-neo-Rep-WDVLIR-Ubipro/intron- Active Yes

[0151] Two truncated inactive neo genes on the same T-DNA were used tomonitor the homologous recombination between T-DNAs in BMS cells. Oneneo gene has a 5′ deletion (5′Δneo), while the second gene has a 3′deletion (3′Δneo), both truncated fragments share a significant regionof overlapping homology comprising 653 bp. Experiments were done ineither the presence or absence of WDV Replicase (Rep). If homologousrecombination occurs, inactive neo genes are restored to produce anactive, full-length neo gene and the cells acquire kanamycin-resistance.

[0152] BMS cells were transformed with the vectors of Table 1 accordingto the Agrobacterium-mediated transformation protocol illustrated inExample 3A. Plasmid P10525 is a positive control transformation vector.Untransformed BMS cells were used as the negative control.

[0153] T-DNA recombinants were identified by the ability to transform E.coli. In order to test for recombinants, DNA was isolated fromuntransformed BMS cells (negative control) and transformed BMS cells.Further, to determine if the T-DNAs could recombine in Agrobacterium,DNA was isolated from the strains used for Agrobacterium-mediatedtransformation of BMS cells.

[0154] DNA was isolated from 2 ml aliquots of Agrobacterium 3 hoursafter acetosyringone induction. Plasmid DNA was isolated using theQiagen DNA mini-prep kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. An aliquot of each sample was adjusted to astandard concentration of 17 ng/μl and used for further analysis. AllDNA samples were stored at −20° C.

[0155] DNA was extracted from BMS cells harvested 7 days afterco-cultivation with Agrobacterium using the DNeasy Plant Mini Kit(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.Briefly, 100 mg of BMS cells were ground to a fine powder in pre-chilledmortars and liquid nitrogen. 400 μl of extraction buffer (buffer AP1)and 4 μl of Rnase A stock (100 mg/ml) were added to the ground cells.Isolated DNA was eluted in either water or buffer AE. DNA concentrationwas estimated using the PicoGreen dsDNA quantitation kit (MolecularProbes, Eugene, Oreg.). An aliquot of each DNA sample was adjusted to 17ng/μl. All DNA samples were stored at −20° C.

[0156] Forty microliters of library-efficiency DH5α E. coli (GibcoBRL)was transformed with 24 ng of DNA from each treatment byelectroporation. The electroporation was performed in a Bio-Rad GenePulser (Bio-Rad, Hercules, Calif.) at 2.5 KV with capacitance set at 25μF, resistance set at 200 ohms and time set at constant using 2 mmcuvettes. Electroporated cells were incubated in 0.6 ml of 2×YT media at37° C. for 30 min. After incubation, 0.2 ml samples were dispensed ontoagar plates containing LB medium supplemented with 0.1 g/L ampicillin.The plates were incubated overnight at 37° C., the number of coloniesper plate was counted. The number of recovered colonies per plate wasaveraged for each treatment.

[0157] Forty colonies were randomly picked and inoculated onto LB agarplates containing kanamycin (0.1 g/L) to screen for homologousrecombinants. The results of this screen are shown in Table 2. TABLE 2Kanamycin resistance generated by homologous recombination of T-DNAAmpicillin-resistant colonies Transformation Total Kan-Resistant % BMSonly 0 0 0 Agro only 77  1/20 5 BMS + Agro 39  0/20 0 BMS + (Agro + Rep)313 65/80 82

[0158] Number of recovered, ampicillin- and kanamycin-resistant coloniesafter E. coli electroporation with DNA isolated from BMS cells only,Agrobacterium only, or BMS cells co-cultivated with Agrobacterium.

[0159] BMS+Agro: DNA from BMS cells co-cultivated with Agrobacteriumcontaining T-DNA without the rep gene (P16823)

[0160] BMS+(Agro+rep): DNA from BMS cells co-cultivated withAgrobacterium containing

[0161] T-DNA (P16821 or P16822) with the rep gene

[0162] The column labeled as “Total” presents the total number ofampicillin-resistant colonies recovered from each particular treatment.The column “Kan-Resistant” presents the fraction of kanamycin-resistantcolonies recovered among the tested ampicillin-resistant colonies.

[0163] These results indicate that homologous recombination did notoccur in the Agrobacterium harboring the T-DNA, it only occurred in thetransformed BMS cells. Further, these results indicate that homologousrecombination occurred only in the presence of replicase (Rep), when Repwas deleted, no kanamycin-resistant colonies were recovered. Once therep gene was provided, 82% of the ampicillin-resistant colonies werealso kanamycin-resistant.

[0164] In order to show that the homologous recombination occurred inthe BMS cells and was not an artifact from the later E. colitransformation, isolated putative recombinant T-DNAs were subjected toexonuclease III treatment and used to transform E. coli as describedabove. The exonuclease degrades linear DNA while circular recombinantsare not affected. No difference in transformation efficiency wasobserved between untreated and exonuclease Ill-treated T-DNAs. Theseresults indicate that homologous recombination occurred in the BMS cellsand was not an artifact of E. coli transformation.

[0165] A restriction digest followed by electrophoretic separation wasused to confirm that the kanamycin-resistant lines contained a restoredfull-length neo gene. A SacII restriction site was located upstream ofthe neo gene and a SphI site was located downstream of the neo gene. Ifthe neo gene is restored via homologous recombination to a full-lengthgene, a SacII/SphI restriction digest yields a band of 1009 bp on anagarose gel. If the truncated neo gene has not been restored tofull-length by recombination, a SacII/SphI restriction digest yields aband of 847 bp on an agarose gel. Control plasmids and DNA from 13kanamycin-resistant and 2 kanamycin-sensitive lines were subjected toSacII/SphI restriction enzyme digestion and agarose gel separation.Results confirm that 11 of the 13 kanamycin-resistant lines had a bandat 1009 bp consistent with the restoration of a full-length neo gene byhomologous recombination in BMS cells, the other two kanamycin resistantlines showed two bands of slightly >1009 bp and ≦847 bp, likelyindicating an additional rearrangement of the recombined neo gene. Bothkanamycin-sensitive lines lacked the presence of the 1009 bp bandindicative of a full-length neo gene.

[0166] PCR analysis was also used to confirm that kanamycin-resistancewas due to the restoration of a full-length neo gene by homologousrecombination.

[0167] B. Functional Replicase is Required to Increase HomologousRecombination

[0168] The pWI-11 vector was the source of the wheat dwarf virus (WDV)initiator protein gene (rep) (Ugaki, M. et al. (1991) Nucl. Acids Res.19:371-377). An NcoI-SacII fragment of this vector containing the repcoding sequence and the short intergenic region (SIR) was subcloned intothe multiple cloning site of pUC19. The long intergenic region (LIR)regulatory element was amplified by PCR to produce a BamHI-NcoIfragment, which was subsequently ligated with the rep NcoI-SphI fragmentand cloned into the BamHI/SphI restriction sites of a gusA expressionvector. This three-fragment ligation produced an expression vectorcontaining gusA and rep, whose expression was controlled by thebi-directional (divergent) promoters within the LIR region. The LIRregion also contained the origin of replication (or) required for vectoramplification in plant cells. Subsequently, the gusA gene was modifiedto include the potato ST LS1 intron (Vancanneyt, G. et al., (1990) Mol.Gen. Genet. 220:245-250). The maize ubi1 intron (Christensen and Quail(1996) Transgenic Res. 5:213-218) containing an FRTI site was insertedbetween the LIR promoter and the gusA coding sequence.

[0169] In order to produce T-DNA vectors, a synthetic FRT1 site (48 bp)was inserted into the multiple cloning site between two T-DNA bordersequences in pSB11 (Ishida, Y. et al. (1996) Nat. Biotech. 14:745-750).The gusA/rep-containing vectors, which included the plasmid backbonewith the ampicillin-resistant gene, were integrated into this site byin-vitro site-specific recombination catalyzed by the FLP protein. Thereaction contained 25 mM Tris-HCI, pH 7.4, 1 mM EDTA, 1 mM DTT, 5%glycerol, 0.1 mM NaCl, 2 μg each of the FRTI-containing vectors to beintegrated, and 1.4 μg of FLP protein in a total volume of 10 μl.Incubation was for 60 min at 30° C. Two microliters of the incubationmixture were used for transformation of library efficiency DH5α. E. colicompetent cells (Cat# 18263-012, Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's specifications. Bacterial colonies were grown at37° C. overnight in spectinomycin-containing (100 mg/L) LB medium andthen transferred into 2 ml of ampicillin-containing (100 mg/L) liquid LBmedium for identification and DNA preparation ofdouble-antibiotic-resistant, co-integrative plasmids.

[0170] Five vectors were generated as shown in Table 3 below. Inexperimental constructs, the gusA gene is separated from its promoter byT-DNA border sequences. Any recombination event within the FRTI sites orthe border sequences will generate replication competent circular T-DNAsin which the recombination marker gene, gusA, is activated. SUGindicates an opposite orientation of the gusA gene in relation to itspromoter on the other end of the T-DNA. Two promoters were used to drivethe expression of gusA, a maize ubiquitin promoter (Upro) was used inthe transformation control vectors (Upro-SUG and Upro-GUS), and v-sensepromoter (Wpro) of WDV was used in the experimental vectors (Wpro-SUG,W-proRep-SUG, and WproRepm-SUG). The v-sense promoter is part of the LIRcompact viral genetic element. This element also contains the viral(+)strand DNA replication origin (ori) and regulatory sequencescontrolling expression of the WDV initiator protein (Rep).

[0171] Tri-parental mating or electroporation was used to integrate thepSB11-based vectors into the super-binary vector pSB1 residing inAgrobacterium tumefaciens strain LBA 4404. Co-integrates were identifiedby double selection of transformed Agrobacterium colonies on mediacontaining spectinomycin and tetracyclin at 100 mg/L each. Restrictionanalysis was used to verify the structural integrity of the super-binaryvectors. TABLE 3 GUS constructs Plasmid Description Rep WproRepm-SUGRB-FRT1/Ubi3′intron-3′gusA-Amp^(r)- No LIR/Repm-Ubi5′intron-FRT1-LBWproRep-SUG RB-FRT1/Ubi3′intron-3′gusA-Amp^(r)- YesLIR/Rep-Ubi5′intron-FRT1-LB Wpro-SUGRB-FRT1/Ubi3′intron-3′gusA-Amp^(r)-LIR- No Ubi5′intron-FRT1-LB Upro-SUGRB-FRT1/Ubi3′intron-3′gusA-Amp^(r)- No Ubipro-Ubi5′intron-FRT1-LB

[0172] TABLE 4 Recovery of T-DNAs Number of colonies/plate Plasmid 3Days 6 Days WproRepm-SUG 28 ± 4 10 ± 8  WproRep-SUG 25 ± 6 170 ± 20 Wpro-SUG 13 ± 1 18 ± 4  Upro-GUS  1 ± 0 0.5 ± 0.7

[0173] BMS cells were transformed with the vectors of Table 3 asdescribed in Example 3A. Circular, recombinant T-DNAs were recoveredfrom total DNA preparations obtained from BMS cells three and six daysafter transformation (see Table 4) and used to transformation of DH5α E.coli as described earlier. In the WproRep-SUG treatment, moreampicillin-resistant colonies were observed using DNA isolated from BMScells six days after transformation compared to DNA isolated three daysafter transformation. No such increase was seen in the Wpro-SUGtreatment, where the vector lacks the initiator protein (rep) gene, orin the WproRepm-SUG treatment, where the C2 open reading frame of theinitiator rep gene was mutated to eliminate replication function. Theseresults indicate that more recombinant T-DNA molecules are produced inBMS cells in the presence of the WDV replicase six days aftertransformation. Since the initiation of T-DNA recombination/replicationrequires accumulation of the rep gene product, the process is apparentlydelayed compared to a direct expression of transformation marker genesin transgenic BMS cells (see also Table 6). TABLE 5 Recovery of T-DNAs+/− FLP Number of colonies/plate 3 Days 6 Days Plasmid −FLP +FLP −FLP+FLP WproRep-SUG 25 ± 6 28 ± 6 170 ± 20 504 ± 107 Wpro-SUG 13 ± 1 20 ± 3 18 ± 4   20 ± 9 

[0174] The circular, recombinant T-DNAs can be formed by site-specificrecombination at the FRTI sites, or by homologous recombination at theT-DNA borders. As analyzed by PCR, in the absence of FLP, junction sitesare generated mostly by the recombination around the border sequences,as indicated by a 661 bp PCR product resulting from border-to-borderrecombination. In the presence of FLP, the size of the predominant PCRproduct is smaller and corresponds to the expected size of theFRT-recombined T-DNA molecules of 307 bp. Generation of these moleculeswas independent of the method of FLP delivery, as the FLP expressionunit was provided on the same T-DNA, delivered by co-transformation, orby a combination of both methods. Further, in treatments with FLP, noPCR amplification signal was observed from the border-to-borderjunction.

[0175] Circular T-DNA molecules recovered 6 days after co-cultivationwere analyzed further. No recombinant T-DNA molecules were recoveredfrom treatments containing only FLP with no Rep. A random sample of 27recombinant T-DNAs was sequenced through the recombination sites toverify that they were generated by site-specific recombination. Of those27 T-DNAs sequenced, 20 T-DNA junction sites were the result ofrecombination events within the two FRTI sites, presumably catalyzed byFLP. TABLE 6 GUS expression GUS expression (nmol MU/min/PCV BMS cells)Days after Transformation Plasmid 1 2 3 4 5 6 7 No DNA 4 6 7 7 7 6 6WproRep-SUG 13 16 11 52 134 395 518 Upro-SUG 6 10 38 37 58 92 52Upro-GUS 8 78 179 242 275 278 277

[0176] The gusA gene separated from its promoter by T-DNA bordersequences produced strong GUS activity in BMS cells co-cultivated withan Agrobacterium strain containing the WproRep-SUG T-DNA (see Table 6).Expression of GUS was delayed by about 1-2 days as compared to the fullyfunctional gusA expression cassette, Upro-GUS, which was used as apositive control. This delay could not be attributed to backgroundactivity since the Upro-SUG control showed only a fraction of the GUSactivity observed in the WproRep-SUG treatment. In addition, no GUSactivity was detectable when the ubiquitin promoter in Upro-GUS wasreplaced with the LIR promoter. The LIR promoter is only activated inthe presence of the initiator Rep protein. These results indicate afunctional gusA gene was generated by a concomitant recombination andreplication of T-DNAs.

[0177] A sample of recombinant T-DNA molecules recovered from treatmentswith the Wpro-SUG vector was sequenced to determine the junction sitesaround the left border. Among 44 randomly selected clones, 33 producedsequencing data. Among them, only two recombinant T-DNAs originated fromrecombination events at the same site, ie. 2 of 33 events had the samejunction sequence. One recombination junction site was identified withinthe FRT sequences sharing a perfect 76 bp homology, but FLP protein wasnot provided in this particular treatment, therefore the recombinationwas based on sequence homology and not produced by the action of asite-specific recombinase. These results indicate the population ofrecombinant T-DNAs generated is likely highly heterogeneous and may notoriginate from a limited number of T-DNA recombination events.

[0178] Sequence analysis of the LB junction sites indicated a variety ofstructural features. While no precise right and left border T-DNAjunctions were identified, two intact RB ends and one LB ends were foundin conjunction with the other modified T-DNA ends. Microhomologiesranging from 1 to 6 bp were common at the crossover sites. Four examplesof filler DNA at the junction sites were found. The sequencing primerwas positioned about 350 bp from the left border, which biased theanalysis towards recovery of left border junction sequences.Nonetheless, 75% of randomly selected clones produced sequencing dataindicating that left border recombination was a preferred mode forgenerating recombinant T-DNAs.

Example 2 Vectors for Plant Transformation

[0179] Vector construction is done using standard molecular biologytechniques. The method of transformation is not critical to theinvention, therefore any method of transformation can be used, andvector construction and/or insert preparation can be modifiedaccordingly.

[0180] A. Introduced Transgene and Targeting Vector

[0181] i. Introduced GUS Transgene

[0182] An Agrobacterium transformation vector was constructed containinga GUS expression cassette between the left and right borders. The GUSexpression cassette comprised 5′UTR::Ubiquitin promoter::maize ubiquitinintron 1::gusA exon 1::gusA intron 1::gusA exon 2::pinIIterminator::3′UTR. The 5′ and 3′ regions each have one SphI site. SphIrestriction enzyme digestion produces a 6.0-6.5 kb DNA fragment. PCRprimers hybridizing to ubi intron 1 and gusA intron 2 amplify a 0.7 kbfragment. The overall structure of the transgene is as follows:

[0183] 5′ ubi pro::ubi intron 1::gusA exon 1::gusA intron 1::gusA exon2::pinII 3′

[0184] ii. GUS Targetinq Vector

[0185] The introduced gusA transgene was used as a target site for agene targeting experiment. The gene targeting vector was containedbetween the left and right border in an Agrobacterium transformationvector. The gene targeting vector was designed to replace gusA exon 1with a bar selectable marker gene which contains a SphI site.Restriction digestion with SphI now results in a 2.3 kb fragment. PCRamplification with the primers directed to introns 1 and 2 generates a1.1 kb fragment. Removal of the gusA exon 1 eliminates GUS expression.

[0186] The structure of the gene targeting vector is as follows:5′LB-ubi pro::ubi intron 1::bar:gusA intron 2::gusA exon2-LIR::Rep::SIR-RB 3′ wherein LIR is the Wheat Dwarf Virus (WDV) longintergenic region containing the promoter and origin of replication; Repis the WDV replicase gene; and SIR is the WDV short intergenic regioncontaining polyadenylation signals.

[0187] B. Gene Targeting System for an Endogenous Genomic Target Site:Acetohydroxy-Acid Synthase (AHAS)

[0188] Point mutations in acetohydroxy-acid synthase (AHAS) can beintroduced to confer either a sulfonylurea or imidazolinone herbicideresistance phenotype in plants.

[0189] i. Tobacco

[0190] There are two genetically unlinked AHAS loci, SURA and SuRB, inNicotiana tabacum, herbicide resistance can be mediated by mutation ateither locus (Chaleff et al. (1986) in Molecular Strategies for CropProtection: UCLA Symposium on Molecular and Cellular Biology,48:415-425, Arntzen and Ryan Eds, John Wiley and Sons, NY; Lee et al.(1988) EMBO J. 7:1241-1248). For example, a sulfonylurea herbicideresistance phenotype can be generated in tobacco by targetedmodification of SuRB to convert Trp 573—Leu 573 (W573L) as described byLee et al. (1990) Plant Cell 2:415-425. The targeting vectors used inLee et al. (supra) can be modified to enhance gene targeting frequencyby the inclusion of an origin of replication (ori) and a replicaseexpression cassette. Using standard vector construction and molecularbiology techniques, gene targeting vector pAGS182BV is modified asfollows:

[0191] 3′RB—5′ΔAHAS—3′ocs::nptII::pnos—3′ocs::Rep::pnos—ori—LB 5′wherein 5′ΔAHAS indicates a 5′ deleted version of the SurB genecontaining the W573L mutation as described by Lee et al. (supra). Theresulting vector will be referenced as pAGS182Bvrep. Vector pAGS180BVcan be modified in a similarway.

[0192] ii. Maize

[0193] Two AHAS genes, AHAS108 and AHAS109, have been reported in maize(Fang et al. (1992) Plant Mol. Biol. 18:1185-1187), herbicide resistancecan be generated by mutation at either locus. For example, asulfonylurea herbicide resistance phenotype can be generated in maize bya targeted modification of AHAS108 to convert Pro 165-Ala 165 (P165A) asdescribed by Lee et al. (1988) EMBO J. 7:1241-1248. An imidazolinoneherbicide resistance phenotype can be generated in maize by a targetedmodification of AHAS108 to convert Ser 621-Asn 621 (S621 N) as describedby Sathasivan et al. (1991) Plant Physiol. 97:1044-1050.

[0194] Using standard vector construction and molecular biologytechniques, targeting vectors can be constructed as follows:

[0195] 5′ LB-ori-ubi::Rep::nos—AHAS108 S621 N-RB 3′

[0196] 5′ LB-ori-ubi::Rep::nos—AHAS108 P165A-RB 3′

[0197] The mutant AHAS genes are not operably linked to a promoter,therefore random integration is unlikely to yield a herbicide resistantphenotype.

[0198] C. Targeting Vectors Introduced by Sexual Crosses

[0199] In order to be maintained for delivery via sexual crosses, thetargeting vector must be integrated into the genome of a plant andexcised after crossing to a second plant. Therefore the targeting vectormust be flanked by excision sequences, for example site-specificrecombination sites or transposon terminal repeats. This exampleoutlines a strategy using a site-specific recombinase.

[0200] The targeting vector is flanked at the 5′ and 3′ ends by directlyrepeated FRT sequences. Adjacent to the 5′ FRT (i.e. directly inside theFRT site) is the Wheat Dwarf Virus replicase gene (the Rep C1:C2sequence). Adjacent to the 3′ FRT site (i.e. directly inside the site)is a promoter, for example the Wheat Dwarf Virus LIR which contains theviral promoter elements and the viral origin of replication (ori). Inthe center of the cassette (i.e. in between the LIR and the replicasegene) is the mutant AHAS sequence (i.e. the target-modifying sequence).This arrangement is shown below:

[0201] 5′FRT-replicase-mutant AHAS-LIR (Promoter & ori)—FRT 3′ Outsidethe targeting vector, but within the T-borders, is a selection cassette,for example UBI::bar::pinil. The transformation cassette is shown below:

[0202] 5′ LB-ubi::bar::pinII-FRT-replicase-mutant AHAS-LIR-FRT3′

Example 3 Transformation

[0203] This example provides methods of plant transformation andregeneration using the polynucleotides of the present invention. Themethod of transformation is not critical to the invention, therefore anymethod of transformation can be used.

[0204] A. Agrobacterium-Mediated Transformation of BMS Cells

[0205]Zea mays Black Mexican Sweet (BMS) cells were propagated inMurashige and Skoog medium containing 4.3 g/L MS salts, 3% sucrose, 2mg/L 2,4-D, 0.1 g/L myoinositol, 0.5 mg/L nicotinic acid, 0.1 mg/Lthiamine-HCL, 0.5 mg/L pyridoxine-HCL, and 2 mg/L glycine, pH 5.6. Thesuspension cultures were shaken at 125 rpm at 25° C. in the dark. Fortransformation, aliquots of cell suspension (5 ml, 0.4 packed cellvolume/ml) were transferred into 50-ml conical tubes and the MS mediumwas replaced with 5 ml of N6 medium (4 g/L N6 basal salts, 6.85%sucrose, 1.5 mg/L 2,4-D, 0.69 g/L L-proline, 0.5 mg/L thiamine-HCI, and1× Eriksson's vitamin mix, pH 5.2) supplemented with acetosyringone at0.1 mM concentration. The same medium (5 ml) was used to re-suspend thepellet of 2.5×10⁸ Agrobacterium cells (centrifuged at 4K rpm for 15 min)that were grown overnight in 30 ml of a minimal medium containing 10.5g/L K₂HPO₄, 4.5 g/L KH₂PO₄, 1 g/L ammonium sulfate, 0.5 g/L sodiumcitrate dihydrate, 1 mM magnesium sulfate, and 0.2% sucrose. The twocell suspensions, BMS cells and Agrobacterium, were combined and placedon a gyratory shaker at 140 rpm for 3 hrs at 27° C. in the dark. Fiftyμl samples of the BMS/Agrobacterium co-cultivation mixtures were placedon dry glass microfiber filters (VWR Scientific Products), andtransferred onto the N6 co-cultivation medium similar to the one usedfor the initial pre-incubations but containing 3% sucrose, 2 mg/L 2,4-D,pH 5.8, and supplemented with 0.3% agar. Plates were incubated in thedark at 27° C. for 24 hrs. Filters were transferred onto the same mediasupplemented with 100 mg/L carbenicilin to eliminate Agrobacterium.

[0206] B. Particle Bombardment Transformation and Regeneration of MaizeCallus

[0207] Immature maize embryos from greenhouse or field grown High typeII donor plants are bombarded with a plasmid or insert containingpolynucleotide of the invention. If the polynucleotide does not includea selectable marker, another plasmid containing a selectable marker genecan be co-precipitated on the particles used for bombardment. Forexample, a plasmid containing the PAT gene (Wohlleben et al. (1988) Gene70:25-37) which confers resistance to the herbicide Bialaphos can beused. Transformation is performed as follows.

[0208] The ears are surface sterilized in 50% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate. These are cultured on 560Lagar medium 4 days prior to bombardment in the dark. Medium 560L is anN6-based medium containing Eriksson's vitamins, thiamine, sucrose,2,4-D, and silver nitrate. The day of bombardment, the embryos aretransferred to 560Y medium for 4 hours and are arranged within the2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L withhigh sucrose concentration).

[0209] The plasmid or insert DNA for transformation is precipitated onto1.1 μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows: 100 μl prepared tungsten particles (0.6 mg) inwater, 20 μl (2 μg) DNA in TrisEDTA buffer (1 μg total), 100 μl 2.5 MCaCl₂, 40 μl 0.1 M spermidine.

[0210] Each reagent is added sequentially to the tungsten particlesuspension. The final mixture is sonicated briefly. After theprecipitation period, the tubes are centrifuged briefly, liquid removed,washed with 500 ml 100% ethanol, and centrifuged again for 30 seconds.Again the liquid is removed, and 60 μl 100% ethanol is added to thefinal tungsten particle pellet. For particle gun bombardment, thetungsten/DNA particles are briefly sonicated and 5 μl spotted onto thecenter of each macrocarrier and allowed to dry about 2 minutes beforebombardment.

[0211] The sample plates are bombarded at a distance of 8 cm from thestopping screen to the tissue, using a DuPont biolistics helium particlegun. All samples receive a single shot at 650 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

[0212] Four to 12 hours post bombardment, the embryos are moved to 560P(a low osmoticum callus initiation medium similar to 560L but with lowersilver nitrate), for 3-7 days, then transferred to 560R selectionmedium, an N6 based medium similar to 560P containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, callus clones are sampled for PCR and activity of thepolynucleotide of interest. Positive lines are transferred to 288Jmedium, an MS-based medium with lower sucrose and hormone levels, toinitiate plant regeneration. Following somatic embryo maturation (2-4weeks), well-developed somatic embryos are transferred to medium forgermination and transferred to the lighted culture room. Approximately7-10 days later, developing plantlets are transferred to medium in tubesfor 7-10 days until plantlets are well established.

[0213] Plants are then transferred to inserts in flats (equivalent to2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to ClassiC™ 600 pots (1.6 gallon) and grown tomaturity. Plants are monitored for expression of the polynucleotide ofinterest.

[0214] C. Agrobacterium-Mediated Transformation and Regeneration ofMaize Callus

[0215] For Agrobacterium-mediated transformation of maize, a genetargeting vector of the present invention is introduced using the methodof Zhao (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326;the contents of which are hereby incorporated by reference).

[0216] Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium containing a polynucleotideof the present invention, where the bacteria are capable of transferringthe nucleotide sequence of interest to at least one cell of at least oneof the immature embryos (step 1: the infection step). In this step theimmature embryos are immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured with theAgrobacterium (step 2: the co-cultivation step). The immature embryosare cultured on solid medium following the infection step. Followingthis co-cultivation period an optional “resting” step is available. Inthis resting step, the embryos are incubated in the presence of at leastone antibiotic known to inhibit the growth of Agrobacterium without theaddition of a selective agent for plant transformants (step 3: restingstep). The immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). The immature embryos are cultured on solid medium with aselective agent resulting in the selective growth of transformed cells.The callus is then regenerated into plants (step 5: the regenerationstep), and calli grown on selective medium are cultured on solid mediumto regenerate the plants.

[0217] D. Transformation of Dicots

[0218] A polynucleotide of the present invention can be introduced intoembryogenic suspension cultures of soybean by particle bombardment usingthe methods as essentially described in Parrott, W. A., L. M. Hoffman,D. F. Hildebrand, E. G. Williams, and G. B. Collins (1989) Plant CellRep. 7:615-617. This method, with modifications, is described below.

[0219] Seed is removed from pods when the cotyledons are between 3 and 5mm in length. The seeds are sterilized in a bleach solution (0.5%) for15 minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced (flat side up) SB1 initiation medium (MS salts, B5 vitamins, 20mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH 5.8). The petri platesare incubated in the light (16 hr day; 75-80 μE) at 26° C. After 4 weeksof incubation the cotyledons are transferred to fresh SB1 medium. Afteran additional two weeks, globular stage somatic embryos that exhibitproliferative areas are excised and transferred to FN Lite liquid medium(Samoylov, V. M., D. M. Tucker, and W. A. Parrott (1998) In Vitro CellDev. Biol.—Plant 34:8-13). About 10 to 12 small clusters of somaticembryos are placed in 250 ml flasks containing 35 ml of SB172 medium.The soybean embryogenic suspension cultures are maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights (20 μE) on a 16:8 hour day/night schedule. Cultures aresub-cultured every two weeks by inoculating approximately 35 mg oftissue into 35 mL of liquid medium.

[0220] Soybean embryogenic suspension cultures are then transformedusing particle gun bombardment (Klein et al. (1987) Nature (London)327:70; U.S. Pat. No. 4,945,050). A BioRad Biolistic™ PDS1000/HEinstrument can be used for these transformations. A selectable markergene, which is used to facilitate soybean transformation, is a chimericgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odellet al. (1985) Nature 313:810-812), the hygromycin phosphotransferasegene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene25:179-188) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0221] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles isthen loaded on each macro carrier disk.

[0222] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. Membrane rupture pressure is set at 1100psi and the chamber is evacuated to a vacuum of 28 inches mercury. Thetissue is placed approximately 8 cm away from the retaining screen, andis bombarded three times. Following bombardment, the tissue is dividedin half and placed back into 35 ml of FN Lite medium.

[0223] Five to seven days after bombardment, the liquid medium isexchanged with fresh medium. Eleven days post bombardment the medium isexchanged with fresh medium containing 50 mg/mL hygromycin. Thisselective medium is refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue will be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue isremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each new lineis treated as an independent transformation event. These suspensions arethen subcultured and maintained as clusters of immature embryos, ortissue is regenerated into whole plants by maturation and germination ofindividual embryos.

[0224] E. DNA Isolatio

[0225] i. DNA Isolation from Callus and Leaf Tissues

[0226] In order to screen putative transformation events for thepresence of the transgene, genomic DNA is extracted from calluses orleaves using a modification of the CTAB (cetyltriethylammonium bromide,Sigma H5882) method described by Stacey and Isaac (1994). Approximately100-200 mg of frozen tissues is ground into powder in liquid nitrogenand homogenized in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA,0.1 M Tris-HCI pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65° C.Homogenized samples are allowed to cool at room temperature for 15 minbefore a single protein extraction with approximately 1 ml 24:1 v/vchloroform:octanol is done. Samples are centrifuged for 7 min at 13,000rpm and the upper layer of supernatant collected using wide-mouthedpipette tips. DNA is precipitated from the supernatant by incubation in95% ethanol on ice for 1 h. DNA threads are spooled onto a glass hook,washed in 75% ethanol containing 0.2 M sodium acetate for 10 min,air-dried for 5 min and resuspended in TE buffer. Five μl RNAse A isadded to the samples and incubated at 37° C. for 1 h.

[0227] For quantification of genomic DNA, gel electrophoresis isperformed using a 0.8% agarose gel in 1×TBE buffer. One microliter ofthe samples are fractionated alongside 200, 400, 600 and 800 ng μl⁻¹¹uncut DNA markers.

Example 4 Gene Targeting

[0228] This example provides methods and constructs used to produce atargeted modification to a polynucleotide integrated in the host genome.The example describes the targeting of a stably introduced transgene, aswell as the targeting of an endogenous gene.

[0229] A. Targeted Modification of a Transgene

[0230] BMS cells were stably transformed with a gusA expression vectoras described in Example 3A. The gusA transgene is described in Example2A, part i. The 5′ and 3′ regions of the transgene each have one SphIsite which result in a 6.0-6.5 kb fragment upon SphI restriction enzymedigestion. PCR primers hybridizing to intron 1 and intron 2 amplify a0.7 kb DNA fragment. GUS expression can be measured by several standardmethods known in the art, such as a quantitative fluorimetric assay asdescribed in (Jefferson et al. (1987) EMBO J. 6:3901-3907).

[0231] The introduced gusA transgene was used as a target formodification. A targeting vector was designed which replaces gusA exon 1with a bar selectable marker gene containing a SphI site. Restrictiondigestion with SphI now results in a 2.3 kb DNA fragment. PCRamplification with the primers directed to introns 1 and 2 generates a1.1 kb band. Removal of the gusA exon 1 eliminates GUS expression. ThegusA target modifying polynucleotide shared a total of about 2 kbhomology with the gusA target.

[0232] Calli transformed with the gusA targeting vector weresequentially screened as follows: bar⁺ calli were selected on Basta.These bar⁺ calli represent all transformation events. Basta-resistantcalli were further screened for GUS activity using a fluorimetric assayto identify putative gene targeting events (see Table 7 below). Randomintegration events should be bar⁺, GUS⁺ while gene targeting eventsshould be bar⁺, GUS⁻. Some GUS⁻ events could be generated by genesilencing, therefore putative gene targeting events, along with controlswere further analyzed by PCR with primers directed to introns 1 and 2.The loss of the 0.7 kb band is diagnostic of a gene targeting event.Cells with randomly integrated targeting vector were GUS⁺, bar⁺, withPCR products of 1.1 kb and 0.7 kb. Cells comprising a gene targetedmodification were GUS⁻, bar⁺, with a PCR product of 1.1 kb only, forexample events CD4 and CF5. Selected events were further evaluated bySouthern analysis of genomic DNA digested with SphI using a gusA exon 2probe confirmed the absence of a ˜6.0-6.5 kb band and the presence of a2.3 kb band in gene targeting events. While not all putative events werefully characterized, of 364 Basta-resistant calli generated 2 genetargeting events were fully confirmed using the process described above.Therefore, the frequency of gene targeting is at least 5.5×10⁻³, whichcorresponds well with observations in Arabidopsis (Puchta et al. (1996)PNAS 93:5055-5060). TABLE 7 Gene targeted knockout of GUS expression GUSexpression (nmol MU/min/PCV BMS cells) Time (min) Cell Line 0 15 28 5183 108 136 BMS control 14 21 22 38 43 43 40 FLG75 27 272 511 873 13741856 2202 CD4 18 28 61 91 119 134 124 CF5 28 42 68 88 116 148 158 AC4 2364 191 302 428 561 678

[0233] B. Targeted Modification of an Endogenous Gene

[0234] Point mutations in acetohydroxy-acid synthase (AHAS) can beintroduced to confer either a sulfonylurea or imidazolinone herbicideresistance phenotype in plants. The following examples describe thetargeted modification of tobacco and maize genes to confer herbicideresistance.

[0235] i. Tobacco

[0236] Transformation, selection, and characterization of AHAS genetargeting events can be done as described in Lee et al. (1990) PlantCell 2:415-425 using the modified vector pAGS182BVrep described inExample 2B, as well as the original control and targeting vectors used.Gene targeting frequency can be measured by comparing the frequency oftargeted modification with vector pAGS182BVrep to the frequency withvector pAGS182BV.

[0237] ii. Maize

[0238] Transformation with AHAS targeting vectors of Example 2B can bedone as described in Example 3C. Selection, and characterization of AHASgene targeting events can be done as described in Zhu et al. (1999) PNAS96:8768-8773. Gene targeting events should exhibit herbicide resistancewhile random integration events should be herbicide sensitive. Genetargeting frequency can be measured by comparing the frequency oftargeted modification with the vectors of Example 2B to the frequencywith control vectors lacking either the origin of replication or thefunctional replicase expression cassette.

Example 5 Crossing-Mediated Gene Targeting

[0239] This example provides methods of gene targeting by sexuallycrossing individual plants. Crossing plants results in gene targetingevents in the developing embryos. The endogenous acetohydroxy-acidsynthase (AHAS; E.C. 4.1.3.18), a key enzyme in the synthesis of thebranched chain amino acids, is targeted to be converted to a mutatedform that imparts resistance to an imidazolinone herbicide.

[0240] In this example, two transgenic plant lines are developed. Thefirst transgenic line comprises a FLP recombinase expression cassette,and the endogenous AHAS gene. The second transgenic line comprises anintegrated AHAS gene targeting vector flanked by FRT-sites. By crossingthese two lines, the targeting vector is excised by FLP recombinasewherein it can generate AHAS gene targeted modification resulting in animidazolinone herbicide resistance phenotype in the progeny.

[0241] A. Recombinase Transgenic Lines

[0242] The FLP-expression cassette is introduced into a maize plantcomprising the endogenous AHAS gene, to produce a transgenic event. Thisconstruct contains a selection cassette (UBI::bar::pinII) and a cassettefor constitutive-expression of a recombinase (UBI::moFLP::pinII) bothwithin an Agrobacterium binary vector. This cassette is transformed intoa maize inbred (for example the Pioneer inbred PHN46) usingAgrobacterium-mediated transformation as described in Example 3C, andBialaphos selection is used to recover transgenic events. Transgenicevents are assessed for single-copy integration using Southern analysis,and further analyzed for FLP activity (see for example WO 99/25841).Single-copy, FLP-active events are regenerated, the plants grown tomaturity, and selfed or outcrossed to produce transgenic seed.

[0243] B. Targeting Vector Transgenic Lines

[0244] The second construct is also an Agrobacterium transformationvector. Inside the T-borders of this construct are the molecularcomponents necessary for crossing-base homologous recombination, thetargeting vector. The targeting vector is described in Example 2C.

[0245] The targeting vector is transformed into immature embryos fromthe PHN46 inbred, and Bialaphos selection is used to recover transgenicevents. The Bialaphos-resistant transformants are screened forsingle-copy integration and these events are regenerated. The resultingplants are selfed or outcrossed to produce transgenic seed.

[0246] C. Crossing and Target Modification

[0247] Using stable transformants from the two transgenic lines, crossesare made between the events produced with the above two constructs.These crosses can be made using TO transgenic plants, or with anyprogeny generation of plants (T1, T2 . . . Tn). For example, T1 seedfrom both transformants is planted and grown to maturity. Upon crossing,the FLP recombinase activity provided by the first transgenic lineresults in excision of the FRT-flanked targeting vector from the copy ofthe genome that came from the second transgenic line. When the FRT sitesrecombine to circularize the cassette, the LIR promoter sequence isjuxtaposed to the replicase gene, resulting in replicase expression. Thecircularized targeting vector replicates, enhancing homologousrecombination between the mutant-AHAS and the endogenous AHAS sequence.The result of this homologous recombination is the targeted modificationof the endogenous AHAS sequence to the mutant form which confersresistance to imidazolinone herbicides.

[0248] Progeny plants containing such as modified AHAS locus arescreened by germinating seedlings on 0.7 μM imazethapyr (AC263, 499, orPursuit, technical grade, American Cyanamid), upon whichherbicide-resistant plants are easily distinguished from wild-typeplants (i.e. with an unaltered AHAS gene). Using this method, it isexpected that the mutant-AHAS herbicide-resistance phenotype will beconferred (via homologous recombination) at much higher frequencies inthe resulting progeny (relative to a non-replicating control targetingvector).

[0249] D. Variations

[0250] Variations on this crossing-based strategy can also beincorporated. Examples include using inducible and/or developmental ortissue-specific promoters to control expression of either therecombinase or the replicase genes, using larger regions of homology(i.e. between the target sequence and the target-modifying sequence inthe two respective plants to be crossed), or controlling replicaseactivity by using variants selected for decreased efficacy. Replicationcan also be controlled by using single replicase component-genes fromgeminiviruses in which replicase functions have evolved in separategenes (for example, using the AL1 gene from the AL1, AL2, AL3 replicasecomplex in Tomato Golden Mosaic Virus, see Hanley-Bowdoin et al. (1990)PNAS (USA) 87(4):1446-1450).

[0251] In another variation, one transgenic line can be produced whichcomprises the integrated targeting vector and a recombinase expressioncassette under control of an inducible promoter. This transgenic linecan be crossed to a non-transgenic line, and recombinase expressioninduced such that the progeny of the cross comprise gene targetedmodifications in a non-transgenic background. Using the gene target ofthe current example, AHAS, these progeny could be easily screened.

[0252] In other variations the targeting vector is flanked by theterminal elements of transposons. In these cases, a transposase isprovided to excise the targeting vector.

[0253] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

What is claimed is:
 1. A method for gene targeting in a eukaryotic hostcell comprising: (a) introducing into the host cell a targeting vectorcomprising a target modifying polynucleotide and an origin ofreplication, wherein the target modifying polynucleotide is capable ofhomologous recombination with a target polynucleotide in the host cell;and (b) providing an appropriate replicase to stimulate homologousrecombination between the target modifying polynucleotide and the targetpolynucleotide to produce a modified target polynucleotide.
 2. Themethod of claim 1 wherein the replicase is provided on an expressioncassette.
 3. The method of claim 1 wherein the targeting vector furthercomprises a replicase polynucleotide operably linked to a promoter. 4.The method of claim 3 wherein the replicase polynucleotide is operablylinked to a constitutive promoter.
 5. The method of claim 3 wherein thereplicase polynucleotide is operably linked to an inducible promoter. 6.The method of claim 1 wherein the eukaryotic host cell is a plant cell.7. The method of claim 6 wherein the plant cell is from a monocot or adicot.
 8. The method of claim 7 wherein the plant cell is selected fromthe group consisting of maize, rice, wheat, oats, barley, sorghum,millet, soybean, canola, Brassica, alfalfa, sunflower, safflower,Arabidopsis, cotton, and tobacco.
 9. A host cell produced by the methodof claim 1, wherein the host cell comprises a modified targetpolynucleotide.
 10. The host cell of claim 9 wherein the host cell is aplant cell.
 11. The plant cell of claim 10 wherein the plant cell isselected from the group consisting of maize, rice, wheat, oats, barley,sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 12. The method of claim 1further comprising generating a whole organism comprising the modifiedtarget polynucleotide.
 13. An eukaryotic organism produced by the methodof claim
 12. 14. The eukaryotic organism of claim 13, wherein theorganism is a plant.
 15. The plant of claim 14, wherein the plant is amonocot or a dicot.
 16. The plant of claim 15, wherein the plant isselected from the group consisting of maize, rice, wheat, oats, barley,sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 17. A seed from the plantof claim 14, wherein the seed comprises a modified targetpolynucleotide.
 18. A method for gene targeting in a eukaryotic hostcell comprising: (a) Introducing into the host cell a targeting vectorcomprising a target modifying polynucleotide and an origin ofreplication, wherein the targeting vector is integrated in a hostgenome, and wherein the targeting vector is flanked by site-specificrecombination sites for excision; and (b) providing a site-specificrecombinase to excise the targeting vector, wherein the excisedtargeting vector forms a nucleic acid capable of replication; and (c)providing an appropriate replicase to stimulate homologous recombinationbetween the target modifying polynucleotide and a target polynucleotideto produce a modified target polynucleotide.
 19. The method of claim 18wherein the replicase is provided on an expression cassette.
 20. Themethod of claim 18 wherein the targeting vector further comprises areplicase polynucleotide operably linked to a promoter.
 21. The methodof claim 20 wherein the replicase polynucleotide is operably linked to aconstitutive promoter.
 22. The method of claim 18 wherein the eukaryotichost cell is a plant cell.
 23. The method of claim 22 wherein the plantcell is from a monocot or a dicot.
 24. The method of claim 23 whereinthe plant cell is selected from the group consisting of maize, rice,wheat, oats, barley, sorghum, millet, soybean, canola, Brassica,alfalfa, sunflower, safflower, Arabidopsis, cotton, and tobacco
 25. Ahost cell comprising a modified target polynucleotide produced by themethod of claim
 18. 26. The host cell of claim 25 wherein the host cellis a plant cell.
 27. The plant cell of claim 26 wherein the plant cellis selected from the group consisting of maize, rice, wheat, oats,barley, sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 28. The method of claim 18further comprising generating a whole organism comprising the modifiedtarget polynucleotide.
 29. An eukaryotic organism produced by the methodof claim
 28. 30. The eukaryotic organism of claim 29 wherein theorganism is a plant.
 31. The plant of claim 30, wherein the plant is amonocot or a dicot.
 32. The plant of claim 31, wherein the plant isselected from the group consisting of maize, rice, wheat, oats, barley,sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 33. A seed from the plantof claim 30, wherein the seed comprises a modified targetpolynucleotide.
 34. A method for gene targeting in a plant comprising:(a) sexually crossing a donor plant and a target plant, wherein thetarget plant comprises a targeting vector comprising a target modifyingpolynucleotide and an origin of replication, wherein the targetmodifying polynucleotide is capable of homologous recombination with atarget polynucleotide in the target plant, and wherein the donor plantcomprises a replicase; and (b) growing the target plant for a timesufficient to produce seed having a modified target polynucleotide. 35.The method of claim 34 wherein the plant is a monocot or dicot.
 36. Themethod of claim 35 wherein the plant is selected from the groupconsisting of maize, rice, wheat, oats, barley, sorghum, millet,soybean, canola, Brassica, alfalfa, sunflower, safflower, Arabidopsis,cotton, and tobacco.
 37. A seed produced by the method of claim 34,wherein the seed comprises a modified target polynucleotide.
 38. Theseed of claim 37, wherein the seed is from a plant selected from thegroup consisting of maize, rice, wheat, oats, barley, sorghum, millet,soybean, canola, Brassica, alfalfa, sunflower, safflower, Arabidopsis,cotton, and tobacco.
 39. A method for gene targeting in a plantcomprising: (a) sexually crossing a donor plant and a target plant,wherein the target plant comprises a targeting vector comprising atarget modifying polynucleotide and an origin of replication, whereinthe targeting vector is flanked by site-specific recombination sites,wherein the target modifying polynucleotide is capable of homologousrecombination with a target polynucleotide in the target plant, whereinthe target plant further comprises a replicase, wherein the donor plantcomprises a site-specific recombinase; and (b) growing the target plantfor a time sufficient to produce seed having a modified targetpolynucleotide.
 40. The method of claim 39 wherein the plant is amonocot or a dicot.
 41. The method of claim 40 wherein the plant isselected from the group consisting of maize, rice, wheat, oats, barley,sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 42. A seed produced by themethod of claim 39, wherein the seed comprises a modified targetpolynucleotide.
 43. The seed of claim 42, wherein the seed is from aplant selected from the group consisting of maize, rice, wheat, oats,barley, sorghum, millet, soybean, canola, Brassica, alfalfa, sunflower,safflower, Arabidopsis, cotton, and tobacco.
 44. A method for genetargeting in a eukaryotic host cell comprising: (a) introducing into thehost cell a targeting vector comprising a transposon comprising a targetmodifying polynucleotide and an origin of replication, wherein thetarget modifying polynucleotide is capable of homologous recombinationwith a target polynucleotide in the host cell; (b) providing anappropriate transposase to excise the targeting vector, wherein theexcised targeting vector form a nucleic acid capable of replication; and(c) providing an appropriate replicase to stimulate homologousrecombination between the target modifying polynucleotide and the targetpolynucleotide to produce a modified target polynucleotide.
 45. Themethod of claim 44 wherein the replicase is provided on an expressioncassette.
 46. The method of claim 44 wherein the targeting vectorfurther comprises a replicase polynucleotide operably linked to apromoter.
 47. The method of claim 46 wherein the replicasepolynucleotide is operably linked to a constitutive promoter.
 48. Themethod of claim 46 wherein the replicase polynucleotide is operablylinked to an inducible promoter.
 49. The method of claim 44 wherein theeukaryotic host cell is a plant cell.
 50. The method of claim 49 whereinthe plant cell is from a monocot or a dicot.
 51. The method of claim 50wherein the plant cell is selected from the group consisting of maize,rice, wheat, oats, barley, sorghum, millet, soybean, canola, Brassica,alfalfa, sunflower, safflower, Arabidopsis, cotton, and tobacco.
 52. Ahost cell produced by the method of claim 44, wherein the host cellcomprises a modified target polynucleotide.
 53. The host cell of claim52 wherein the host cell is a plant cell.
 54. The plant cell of claim 53wherein the plant cell is selected from the group consisting of maize,rice, wheat, oats, barley, sorghum, millet, soybean, canola, Brassica,alfalfa, sunflower, safflower, Arabidopsis, cotton, and tobacco.
 55. Themethod of claim 44 further comprising generating a whole organismcomprising the modified target polynucleotide.
 56. An eukaryoticorganism produced by the method of claim
 55. 57. The eukaryotic organismof claim 56, wherein the organism is a plant.
 58. The plant of claim 57wherein the plant is a monocot or a dicot.
 59. The plant of claim 58,wherein the plant is selected from the group consisting of maize, rice,wheat, oats, barley, sorghum, millet, soybean, canola, Brassica,alfalfa, sunflower, safflower, Arabidopsis, cotton, and tobacco.
 60. Aseed from the plant of claim 57, wherein the seed comprises a modifiedtarget polynucleotide.
 61. An isolated polynucleotide comprising atargeting vector, wherein the targeting vector comprises a targetmodifying polynucleotide and an origin of replication, wherein thetarget modifying polynucleotide is capable of homologous recombinationwith a target polynucleotide in a eukaryotic host cell.
 62. The isolatedpolynucleotide of claim 61, wherein the targeting vector furthercomprises a replicase polynucleotide operably linked to a promoter. 63.The isolated polynucleotide of claim 61, wherein the targeting vector isflanked by directly repeated site-specific recombination sites, whereinthe integrated targeting vector is excised in the presence of anappropriate recombinase.
 64. The isolated polynucleotide of claim 61,wherein the targeting vector comprises a transposon, wherein anintegrated targeting vector is excised in the presence of an appropriatetransposase.